Engineered antibodies and methods of treatment

ABSTRACT

The invention is related to CD20 antibodies with improved characteristics. Some embodiments describe antibodies comprising a mouse IgG2; a human IgG1, IgA1 or IgA2 constant region and a variable domain that can bind the epitope “EPANpSEK” on human CD20 expressed on Ramos cells and which antibody has an increased PCD functionality when compared to Rituximab with a constant region of the same isotype.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/329,726, filed Feb. 28, 2019, which is a national phase entry under35 U.S.C. § 371 of International Patent Application PCT/NL2017/050581,filed Sep. 1, 2017, designating the United States of America andpublished in English as International Patent Publication WO 2018/044172A1 on Mar. 8, 2018, which claims the benefit under Article 8 of thePatent Cooperation Treaty to European Patent Application Serial No.16186850.0, filed Sep. 1, 2016, all of which are hereby incorporated byreference in their entirety.

TECHNICAL FIELD

The disclosure relates to the field of antibodies. In particular, itrelates to antibodies that bind CD20. It further relates to the use ofCD20 antibodies in medical and detection methods. The disclosure furtherrelates to cells, nucleic acid molecules and methods for the productionof the antibodies.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 3, 2022, isnamed 199828-702301_SL.txt and is 28,672 bytes in size.

BACKGROUND

Three CD20 mAbs have been approved for the treatment of various subtypesof Non-Hodgkin's lymphomas and leukemias (NHL). Rituximab (RTX), thefirst mAb on the market, has significantly improved survival of patientswhen given in combination with chemotherapy regimens.⁽¹⁻⁵⁾ Ofatumumab(OFA) was selected based on its ability to activate the classicalcomplement pathway, leading to membrane disruption andcomplement-dependent cytotoxicity (CDC). Clinical trials highlighted itsemerging potential in combination therapies and maintenance of e.g.,chronic lymphocytic leukemia (CLL) patients.^((6,7)) Obinutuzumab (OBZ;GA101), a CD20 mAb with enhanced FcγRIII binding and direct programmedcell death (PCD) induction capacity, has been approved for first-linetreatment of CLL⁽²⁰⁾ and RTX refractory follicular NHL.⁽⁹⁾

In vitro, CD20 mAbs can induce antibody-dependent cell-mediatedcytotoxicity/phagocytosis (ADCC/ADCP), CDC or PCD. Depending on theirmechanism-of-actions (MoA), CD20 mAbs are grouped into Type I and TypeII. Both Type I and II mAbs elicit ADCC Type I mAbs, including RTX andOFA, relocate CD20 into lipid rafts and efficiently activate thecomplement system.⁽¹⁰⁾ Type II mAbs induce PCD via a caspase-independentpathway.⁽¹¹⁾ The only described Type II mAbs are OBZ,⁽¹²⁾ B1,⁽¹³⁾ and11B8.⁽¹⁴⁾ Interestingly, RTX was shown to induce PCD via the samepathway in lymphoma cell lines and primary CLL cells, but to a lowerextent than Type II mAbs.⁽¹⁵⁾ Most recently, a CD20 mAb displaying TypeI and Type 1H characteristics in vitro was described.⁽¹⁶⁾

In literature, three features were suggested to govern Type I/Hclassification: (a) the epitope, (b) binding kinetics and (c) residueswithin the elbow-hinge angle determining region of the VH chainframework.

Human CD20 comprises a small (residue 72-80) and larger (residue140-186) extracellular loop. The epitope of RTX is located on the largerloop with ¹⁷⁰ANPS¹⁷³ representing the core binding region.⁽¹⁷⁾Mutagenesis experiments confirmed N171 to be an important residue forRTX.⁽¹⁸⁾ For RTX and another panel of Type I mouse CD20 mAbs A170 andP172 were determined to be important.^((19, 20)) Although Type II mAbsOBZ and B1 have an overlapping epitope⁽¹⁷⁰ ANPSEKNSP¹⁷⁸ (SEQ ID NO: 15))with RTX, residues 176-178 contribute most to the binding.⁽¹⁸⁾ Incontrast, the epitope of the Type I mAb OFA and Type II mAb 11B8 iscomprised by residues on the small and larger loop.⁽²⁰⁾ As OFArecognizes this unique epitope and efficiently activates complement, itwas suggested that the membrane-proximal binding results in a beneficialorientation of the available Fc-fragments allowing better complementdeposition. Additionally, Ab kinetics were proposed to correlate withthe CDC activity of CD20 mAbs, as the strong complement inducer OFAdissociates significantly more slowly from CD20 than RTX with anintermediate CDC capacity.⁽¹⁴⁾ Further evidence supporting thecontribution of a slower off-rate to better CDC induction comes fromstudies with the CD20 mAb veltuzumab.⁽²¹⁾

Next to antigen binding properties, a structural Ab feature wassuggested. During the humanization of BLy-1 (Type I) to OBZ (Type II) aL11V mutation was introduced in the VH chain framework. The reversemutation in OBZ resulted in loss of PCD induction.⁽¹²⁾ Modeling of RTXand OBZ indicated that the L11V mutation results in a wider Abelbow-hinge angle for OBZ (167°) compared to RTX (140°).⁽¹⁸⁾

Although various CD20 antibodies are known there is still little knownabout the properties that determine the mechanism-of-action of CD20mAbs.

BRIEF SUMMARY

The disclosure shows novel CD20 antibodies. All antibodies display TypeI characteristics whereas some of these also display Type IIcharacteristics. It was found that neither the epitope, nor the off-rateare by themselves is enough to predict whether a CD20 antibody exhibitsa Type I, a Type II or Type I/II activity.

This disclosure further provides CD20 antibodies with a human IgAconstant region. IgA is the second most prominent antibody in blood,after IgG, and the predominant Ab at the mucosa The monomeric version ofIgA is mostly found in serum, whereas polymeric IgA is produced atmucosal sites. The 2 Ab subclasses, IgA1 and IgA2, differ structurallyin their hinge regions, which is 13 amino acids longer for IgA1 comparedto IgA2. This might enable an improved reach for antigens that aredistant, but at the same time makes it more prone to degradation byproteases.⁽³⁶⁾ Furthermore, the hinge region of IgA1 Abs carries severalO-linked glycosylation sites, which are absent in IgA2 Abs. IgA2 existsas 3 allotypes; IgA2(m1) which has 2 additional N-linked glycosylationsites compared with IgA1, and IgA2(m2) and IgA2(n), which have 3additional N-linked glycosylation sites. Contrary to IgG, IgA is a weakactivator of the classical complement pathway as it cannot bind C1q.⁽³⁷⁾However, IgA mAbs have been shown to activate the complement systemthrough the lectin pathway, as the carbohydrate recognition domain (CRD)of mannan-binding lectin (MBL) can bind to IgA.⁽³⁸⁾

IgA engages immune effector cells by binding to the FcαRI (CD89), whichis expressed on cells of the myeloid lineage: neutrophils, monocytes,different macrophage populations and eosinophils.⁽³⁹⁾ Expression on invitro generated dendritic cells was shown,^((40,41)) but remainscontroversial. Neutrophils express high levels of FcαRI, whilemacrophages have lower expression.⁽⁴²⁾ In ADCC assays with IgA mAbstargeting solid tumor targets, neutrophils have been shown toefficiently eradicate tumor cells.⁽⁴³⁻⁴⁶⁾ In contrast, IgG1 mAbs wereless able to engage this effector cell population.Monocyte/macrophage-mediated tumor cell killing was shown to becomparable between IgA and IgG mAbs.⁽⁴³⁾ Next to the activatingFcγRIIIa, macrophages also express the inhibitory FcγRIIb. It has beenshown that the presence of FcγRIIb reduces mAb activity.⁽⁴⁷⁾ For IgA, noinhibitory receptor has been described yet. The knowledge on IgA mAbtargeting tumor-associated antigens has increased significantly over thelast few years. Several bottlenecks faced a few years ago are nowovercome, and are currently able to produce and purify sufficientamounts of monomeric IgA mAbs for in vitro and in vivo testing. Micelack a receptor for IgA, therefore the generation of human FcαRItransgenic now allows in vivo testing.⁽⁴⁸⁾ Boross and colleagueseventually showed in an immunocompetent tumor model the great potentialof IgA mAbs in a therapeutic setting.⁽⁴⁴⁾ The majority of IgA mAbsstudied so far are targeting HER2 or EGFR, antigens expressed on solidtumors. Only one study has looked at the potential of monomeric IgA-CD20mAbs.⁽⁴⁹⁾ Complement-mediated tumor cell killing was demonstrated torely on weak indirect activation of the classical pathway and morepronounced direct activation of the alternative pathway. With a passiveimmunization strategy in FcαRI transgenic mice, a good protectionagainst tumor development with monomeric IgA2-CD20 mAbs was achieved.However, therapeutic in vivo testing for IgA-CD20 mAbs has not yet beenperformed. Further, a direct comparison of IgA1 and IgA2 mAbs, inparticular, with respect to their complement activation properties islacking. The disclosure describes unique IgA1- and IgA2-CD20 mAbs.

The disclosure provides an antibody comprising a mouse IgG2; a humanIgG1, IgA1 or IgA2 constant region and a variable domain that can bindthe epitope “EPANpSEK” (one letter code for Glu Pro Ala Asn Pro Ser GluLys (SEQ ID NO:31)) on human CD20 expressed on Ramos cells and whichantibody has an increased PCD functionality when compared to Rituximabwith a constant region of the same isotype.

The disclosure also provides an antibody that can bind to anextracellular part of human CD20 expressed on Ramos cells comprising avariable domain with a heavy chain variable region and a light chainvariable region, wherein the heavy chain variable region comprises aCDR3 region with the sequence SNSYGSTYWYFDV (SEQ ID NO:21).

The disclosure further provides an antibody comprising a mouse IgG2; ahuman IgG1, IgA1 or IgA2 constant region and a variable domain that canbind the epitope “EPANPsEK” (SEQ ID NO:31) on human CD20 expressed onRamos cells and which antibody has an increased ADCC functionality whencompared to Rituximab with a constant region of the same isotype.

Also provided is an antibody that can bind to an extracellular part ofhuman CD2 expressed on Ramos cells, the antibody comprising a variabledomain with a heavy chain variable region and a light chain variableregion, wherein the heavy chain variable region comprises a CDR3 regionwith the sequence YYYGSSYGAMDY (SEQ ID NO:22).

Further provided is an antibody comprising a mouse IgG2; a human IgG1,IgA1 or IgA2 constant region and a variable domain that can bind theepitope “EPANpsEK” (SEQ ID NO:31) on human CD20 expressed on Ramos cellsand which antibody has an increased CDC functionality when compared toRituximab with a constant region of the same isotype.

Also provided is an antibody that can bind to an extracellular part ofhuman CD20 expressed on Ramos cells comprising a variable domain with aheavy chain variable region and a light chain variable region, whereinthe heavy chain variable region comprises a CDR3 region with thesequence TYYYGSSPYWSFDV (SEQ ID NO:23).

Also provided is an antibody that can bind to an extracellular part ofhuman CD20 expressed on Ramos cells comprising a variable domain with aheavy chain variable region and a light chain variable region, whereinthe heavy chain variable region comprises a CDR3 region with thesequence SRLFDSSYGWYFDV (SEQ ID NO:24).

Further provided is an antibody comprising a mouse IgG2; a human IgG1,IgA1 or IgA2 constant region and a variable domain that can bind theepitope “EPANpSEK” (SEQ ID NO:31) on human CD20 expressed on Ramos cellsand which antibody has an increased CDC and/or increased ADCCfunctionality when compared to Rituximab with a constant region of thesame isotype.

Also provided is an antibody that can bind to an extracellular part ofhuman CD20 expressed on Ramos cells comprising a variable domain with aheavy chain variable region and a light chain variable region, whereinthe heavy chain variable region comprises a CDR3 region with thesequence SAYYGSNVWFFDV (SEQ ID NO:25).

Further provided are antibodies as described herein for use in thetreatment of a disease in an individual.

Also provided are antibodies as described herein for use in thetreatment of a disease that involves too many B cells, overactive Bcells, and/or dysfunctional B cells.

Also provided are antibodies as described herein for use in thetreatment of a CD20 positive neoplasm such as a CD20 positive B-celllymphoma; hairy cell leukemia; B-cell chronic lymphocytic leukemia, ormelanoma.

Also provided are methods for the treatment of an individual that has adisease that involves too many B cells, overactive B cells, and/ordysfunctional B cells comprising administering to the individual in needthereof an antibody as described herein.

Also provided are methods for the treatment of an individual that has aCD20 positive neoplasm such as a CD20 positive B-cell lymphoma; hairycell leukemia; B-cell chronic lymphocytic leukemia, or melanomacomprising administering to the individual in need thereof an antibodyas described herein.

Also provided are methods for the treatment of children with B-cellmalignancies and pediatric leukemia patients that have a B-cell diseaseafter stem cell transplantation. In pediatric patients, long-termadverse effects of rituximab are noted: permanent depletion of B cellsand inability of naïve B cells to switch to memory B cells, resulting inlife-long immunoglobulin depletion. The long persistence of IgG in thebody is probably accountable. An IgA antibody described herein has astronger ADCC function when compared to an IgG antibody comprising thesame variable domain. This is apparent when a B-cell-specific marker isanalyzed that is not subject to trogocytosis upon incubation of the cellwith a CD20 antibody. An IgA antibody described herein has a shorthalf-life when compared to an IgG antibody comprising the same variabledomain. The IgA antibodies as described herein cause fewer side effectswhen compared to an IgG antibody with the same variable domain. Inshort, the IgA antibodies of the disclosure facilitate an effective hitbut are also cleared fast enough to allow a good recovery of the B-cellrepertoire. This is particularly helpful in preserving the B-cellrepertoire following recovery from the treatment, particularly in thementioned leukemia patients that have a B-cell disease after stem celltransplantation treated with an antibody of the disclosure and pediatricpatients after B-cell depletion with an antibody of the disclosure.

The disclosure further provides a variable domain comprising the aminoacid sequence of the heavy and light chain variable regions of SEQ IDNOS:1 and 2, each with 0-5 amino acid insertions, deletions,substitutions, additions or a combination thereof at one or morepositions other than positions of the amino acids that constitute theCDR1, CDR2 and CDR3 regions

The disclosure further provides a variable domain comprising the aminoacid sequence of the heavy and light chain variable regions of SEQ IDNOS:7 and 8 each with 0-5 amino acid insertions, deletions,substitutions, additions or a combination thereof at one or morepositions other than positions of the amino acids that constitute theCDR1, CDR2 and CDR3 regions.

The disclosure further provides a variable domain comprising the aminoacid sequence of the heavy and light chain variable regions of SEQ IDNOS: 9 and 10 each with 0-5 amino acid insertions, deletions,substitutions, additions or a combination thereof at one or morepositions other than positions of the amino acids that constitute theCDR1, CDR2 and CDR3 regions.

The disclosure further provides a variable domain comprising the aminoacid sequence of the heavy and light chain variable regions of SEQ IDNOS:11 and 12 each with 0-5 amino acid insertions, deletions,substitutions, additions or a combination thereof at one or morepositions other than positions of the amino acids that constitute theCDR1, CDR2 and CDR3 regions.

The disclosure further provides a variable domain comprising the aminoacid sequence of the heavy and light chain variable regions of SEQ IDNOS:13 and 14 each with 0-5 amino acid insertions, deletions,substitutions, additions or a combination thereof at one or morepositions other than positions of the amino acids that constitute theCDR1, CDR2 and CDR3 regions. Further provided is an antibody comprisinga variable domain as specified herein.

Further provided is

-   -   a nucleic acid molecule that codes for a heavy chain or light        chain of an antibody as described herein;    -   a nucleic acid molecule that codes for the CDR3 of a heavy or        light chains of an antibody as described herein;    -   a nucleic acid molecule that codes for a CDR1, CDR2 and CDR3 of        the heavy or light chain of an antibody as described herein; and    -   a nucleic acid molecule that codes for a variable region of a        heavy chain or of a light chain of an antibody as described        herein.

Further provided is a cell that comprises nucleic acid that codes for anantibody as described herein

Further provided are means and methods for the production of an antibodyas described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1.1 and 1.2 . Most new CD20 mAbs have unique heavy and light chainsequences. Sequences coding for the variable region of the (A) heavychain and (B) light chain were aligned and compared with commerciallyavailable CD20 mAbs by calculating the average distance using % identity(PID). The right panel is a snapshot of the tree on the left.

FIGS. 2A-2C. As mouse antibodies, all new CD20 mouse IgG mAbs exhibitType I characteristics. (FIG. 2A) PCD: EL4-CD20 cells incubated for 24hours with 1 μg/mL CD20 mAbs in the absence or presence of cross-linkingAb. PCD determined by 7-AAD⁺/AnnexinV-PE⁺ staining (mean+SEM). (FIG. 2B)ADCC: Specific lysis of Daudi cells in a chromium release assay withPBMCs as effector cells (E:T=100:1) at 1 μg/mL CD20 mAb (mean+SEM).(FIG. 2C) CDC by new CD20 mAbs was determined at 10 and 1 μg/mL mAb in15.5% human serum and detected by 7-AAD staining((value_(sample)−mean_(medium))+SEM). mIgG2a-CD20 mAbs B1, mRTX, andm7D8 in grey were taken along as controls. mlgG2c mAbs in pink andmIgG2b mAbs in blue.

FIGS. 3A, 3B. New mIgG2c-CD20 mAbs display varying CDC properties.Induction of complement-dependent cytotoxicity by (FIG. 3A) all newmIgG2c-CD20 mAbs on Daudi cells and (FIG. 3B) m1, m2 and m7D8 on Ramosand Raji cells. mIgG2a-CD20 mAbs B1 and m7D8 were taken along ascontrols. Cells were incubated with indicated mAb concentrations and15.5% human serum. Cytotoxicity was determined by 7-AAD staining((value_(sample)−mean_(medium))+SEM).

FIGS. 4A-4C. Binding site on CD20 is different among new CD20 mAbs.(FIG. 4A) Epitope mapping using the circular peptideYNCEPANPSEKNSPSTQYCYS (SEQ ID NO:16) resulted in identification of theepitope of m1 (left) but only marginally of m2 (right). Binding of 1μg/mL mAb to the peptide and mutants with each amino acid replaced withall other available (positional scan; excluding cysteine) was determinedby ELISA. Grey line and shaded area represents WT binding A SEM. Resultsare displayed with Tukey-whiskers. (FIG. 4B) Rough epitope mapping withCD20 WT or CD20 mutant (KDD=T159K/N163D/N166D, A×P=A170S/P172S). HEK293Fcells were transfected with plasmids and binding of mAbs (5 μg/mL) toCD20 was measured by FACS. Binding was compared to Type I CD20 mAbs(m7D8 and mRTX) and Type II mAbs (B1 and m11BS). (FIG. 4C) Determinationof residues crucial for CD20 mAb binding. Data are represented as % ofbest binder. Coloring according to binding compared to best binder (darkgrey: 0-20/=loss of binding; grey: 21-70%=intermediate binding; lightgrey: 71-100/full binding).

FIGS. 5A-5C. New CD20 mAbs with distinct kinetics. Real-time binding anddissociation curve to SKBR3-CD20 cells using Ligand Tracer Green.Association of 10 nM FITC-labeled CD20 mAbs was monitored for 1 hourbefore following the dissociation for 3 hours in the presence of (FIG.5A) RPMI culture medium (non-competitive) or (FIG. 5B) 100 nM unlabeledCD20 mAbs (competitive). (FIG. 5C) Comparison of dissociation rateconstants under non-competitive and competitive conditions determined by1:1 Fitting model.

FIG. 6 . Chimerization can alter PCD activity. Induction of homotypicaggregation (pictures) and PCD by IgG1-CD20-1 and IgG1-CD20-2 mAbs (1μg/mL) with or without theL11V mutation in the absence (light grey bar)or presence (dark grey bar) of cross-linking Ab (for IgG1, a-human IgG:20 μg/mL; for B1, a-mouse IgG: 50 μg/mL). B1 and OFA as positive andnegative control, respectively. Induction of cell death was determinedby 7-AAD and AnnexinV-PE staining (mean+SEM).

FIGS. 7A, 7B. Differences in in vivo efficacy of m1gG2c-CD20 mAbs.C57BL/6 mice (4-6 mice/group) were injected intraperitoneally with 5×105CellTraceViolet labeled EL4-CD20 cells 16 hours prior to mAb or PBStreatment. The anti-tumor response was evaluated 24 hours later bydetermining the amount of remaining tumor cells in the peritoneal lavagewith TruCount tubes. (FIG. 7A) mAb titration of m1, m2 and m7DS(median+interquartile range). (FIG. 7B) Anti-tumor response by 1 μgmIgG2c mAb, represented as % of PBS (median+interquartile range). 1 μgm7D8 (mIgG2a) as positive control. 2 separate experiments as indicatedby dashed line.

FIG. 8 . New chimeric IgG1-CD20 mAbs bind to CD20-expressing Daudicells. Binding was determined by FACS after incubation of Daudi cellswith a dilution series of mAb. RTX and OFA were included as positivecontrols, and Trastuzumab as isotype control.

FIGS. 9A-9G. Analysis of in vitro efficacy of new chimeric IgG1-CD20mAbs. (FIG. 9A) ADCC: Specific lysis of Daudi cells in a chromiumrelease assay with PBMCs as effector cells (E:T=50:1) over a wider mAbconcentration range (mean±SEM). (FIG. 9B) CDC of new CD20 mAbsdetermined in 15.5% human serum and detected by 7-AAD staining((value_(sample)−mean_(medium))±SEM). (FIG. 9C) PCD: EL4-CD20 cellsincubated for 24 hours with 1 μg/mL CD20 mAbs in the absence or presenceof 20 μg/mL cross-linking Ab. PCD was determined by 7-AAD/AnnexinV-PEstaining (mean+SEM). B1 (mIgG2a-CD20 mAb) is a positive control for PCD((value_(sample)−mean_(medium))±SEM). In all assays RTX and OFA wereincluded as positive controls, and TRA as isotype control. PCD inductionon (FIGS. 9D, 9E) Ramos cells and (FIGS. 9F, 9G) Daudi cells byIgG1-CD20-1 without or with the L11V mutation (10 μg/ml). Induction ofcell death was determined by (FIGS. 9D, 9F) 7-AAD/AnnexinVPE stainingand (FIGS. 9E, 9G) DiOC6/TO-PRO-3 staining (mean+SEM). 11B8 and OFA weretaken along as positive and negative control, respectively. Results arerepresentative of 3 separate assays. *p<0.05; **p<0.01; ***p<0.001, byone way ANOVA followed by Bonferroni posthoc analysis.

FIGS. 10A, 10B. Disappearance of CD19+ viable B cells in autologoussetting. B-cell depletion by CD20 mAbs was determined by incubating mAbsfor 1 hour at 37° C. with unprocessed blood from healthy donors. (FIG.10A) FACS plots showing disappearance of viable B cells upon incubationwith mAb (here IgG1-CD20-1) compared to PBS control. (FIG. 10B) B-celldepletion mediated by CD20 mAbs determined over a broader concentrationrange (mean+SEM). RTX and OFA were included as positive controls, andTRA as isotype control.

FIG. 11 . In vivo efficacy of IgG1-CD20 mAbs. C57BL/6 mice (6mice/group) were injected intraperitoneally with 5×10⁵ CellTraceVioletlabeled EL4-CD20 cells 16 hours prior to mAb (10 μg) or PBS treatment.The anti-tumor response was evaluated 24 hours later by determining theamount of remaining tumor cells in the peritoneal lavage with TruCounttubes (median interquartile range).

FIGS. 12A-12C: Production of IgA-CD20 mAbs. (FIG. 12A) Test transfectionof HEK293F cells to determine optimal ratio between HC, LC andpAdvantage coding plasmids. Concentrations of produced IgA-CD20 mAbswere measured by an IgA-specific ELISA. Large scale produced IgA-CD20mAbs were purified by (FIG. 12B) anti-human kappa affinitychromatography, followed by (FIG. 12C) size-exclusion chromatography toseparate the full size antibody from loose light chains and aggregates.Representative graphs are displayed.

FIG. 13 : Binding of IgA-CD20 mAbs to CD20-expressing Ramos cells.Binding was determined by FACS after incubation of Ramos cells with adilution series of mAb.

FIG. 14 : Variable extent of programmed cell death induction by IgA-CD20mAbs. EL4-CD20 cells were incubated for 24 hours with 1 μg/mL mAb in theabsence and presence of cross-linking Ab (B1: 50 μg/mL; IgG1 and IgAmAbs: 20 μg/mL). The degree of cell death was determined as the sum ofAnnexinV⁺ and AnnexinV⁺/7AAD⁺ cells (value_(sample)−mean_(medium)+SEM).B1 (mIgG2a-CD20 mAb) and RTX were taken along as positive controls andOFA as a negative control.

FIGS. 15A-15C: Complement-dependent cytotoxicity induced by IgA-CD20mAbs. Target cells were incubated for indicated time with 15.5% pooledhuman serum and degree of CDC was determined as % 7AAD⁺ cells. (FIGS.15A, 15B) Time dependency of complement induction by IgA-CD20 mAbs.Complement-mediated lysis of (FIG. 15A) Daudi and (FIG. 15B) Ramos cellsincubated for 15, 60, 240, and 360 minutes in the presence of 10 μg/mLmAb. (FIG. 15C) The degree of complement-mediated lysis of Ramos cellsafter 15 minutes incubation with 10 μg/mL mAb was inhibited bypre-treatment of the complement source with the indicated inhibitors(heat inactivated serum; excess of eculizumab; EDTA+MgCl₂). Results areshown as (value_(sample)−mean_(medium))+SEM.

FIGS. 16A-16C: B-cell depletion assay in autologous setting. Whole bloodleukocytes were incubated with CD20 mAbs for 3 hours at 37° C. Analysiswas performed on FSC/SSC lymphocyte gate from which CD3, CD14, CD56 andCD11b positive cells were excluded. CD19 was used as B-cell marker.(FIG. 16A) Analysis of CD19 expression on CD19-cells (B cells). (FIG.16B) Number of CD19⁺ events found in lymphocyte gate. (FIG. 16C) Numberof CD19⁺ cells found in lymphocyte gate after background (no antibody)subtraction.

FIG. 17 . Anti-CD20 IgA mediates efficient tumor cell lysis of CD20targets. ADCC as measured by the release of ⁵¹Cr from EL4-CD20-Luc2cells using (Panel A) PBMC, E:T=100:1, or (Panel B) PMN, E:T=40:1, aseffector cells and the indicated antibody concentrations. Anti-CD20IgA's contain the same variable regions as rituximab.

FIG. 18 . Amino acid sequence of various I and VL chains. CDR sequencesare underlined from left to right in the sequence CDR1, CDR2 and CDR3.

The positions of the CDRs in the VH were determined using the followingcriteria:

CDR-H1

Start—Approx residue 26 (always 4 after a CYS) [Chothia/AbM defintion].Kabat definition starts 5 residues later. Residues before alwaysCYS-XXX-XXX-XXX. Residues after always a TRP. Typically TRP-VAL, butalso, TRP-ILE, TRP-ALA. Length 10 to 12 residues (AbM definition)Chothia definition excludes the last 4 residues.

CDR-H2

Start—always 15 residues after the end of Kabat/AbM definition ofCDR-H1. Residues before typically LEU-GLU-TRP-ILE-GLY (SEQ ID NO:17),but a number of variations. Residues afterLYS/ARG-LEU/ILE/VAL/PHE/THR/ALA-THR/SER/ILE/ALA. Length Kabat definition16 to 19 residues.

CDR-H3

Start—always 33 residues after end of CDR-H2 (always 2 after a CYS).Residues before always CYS-XXX-XXX (typically CYS-ALA-ARG). Residuesafter always TRP-GLY-XXX-GLY (SEQ ID NO:18). Length 3 to 25(!) residues.

The positions of the CDRs in the VL were determined using the followingcriteria:

CDR-L1

Start—Approx residue 24. Residue before is always a Cys. Residue afteris always a Trp. Typically TRP-TYR-GLN, but also, TRP-LEU-GLN,TRP-PHE-GLN, TRP-TYR-LEU. Length 10 to 17 residues.

CDR-L2

Start—always 16 residues after the end of L1. Residues before generallyILE-TYR, but also, VAL-TYR, ILE-LYS, ILE-PHE. Length always 7 residues.

CDR-L3

Start—always 33 residues after end of L2. Residue before is always Cys.Residues after always PHE-GLY-XXX-GLY (SEQ ID NO:19). Length 7 to 11residues.

FIG. 19 . Binding of mIgG-CD20 mAbs to Daudi cells.

FIG. 20 . No induction of HA of EL4-CD20 cells by new CD20 mAbs (1μg/mL) (20× magnification). Cross-linking Ab (50 μg/mL) was added aspositive control conditions B1 as positive control (Type II).

FIGS. 21A, 21B. Epitope mapping using the linear peptide NSPSTQYGPANPSEK(SEQ ID NO:20) resulted in identification of aa contributing to bindingof (FIG. 21A) m1 but not (FIG. 21B) m2. Binding of 1 μg/mL mAb to thepeptide and corresponding mutants with each aa replaced with all otheravailable (positional scan; excluding cysteine) was determined by ELISA.Grey line and shaded area=WT binding SEM; Results are displayed withTukey-whiskers.

FIGS. 22A-22D. (FIGS. 22A, 22B) FACS based dissociation analysis. Daudicells were stained with 10 μg/mL Alexa647-labeled CD20 mAb. Cells wereleft in (FIG. 22A) RPMI culture medium (non-competitive) or (FIG. 22B)the presence of a 10-fold excess of unlabeled mAb (100 μg/mL;competitive). At indicated time points the level of cell-bound mAb wasdetermined. (FIG. 22C, FIG. 22D) Real-time binding and dissociationcurve to SKBR3-CD20 cells using the Ligand Tracer technology.Association of 10 nM FITC-labeled CD20 mAbs was monitored for 4 hoursbefore following the dissociation for 8 hours in the presence of (FIG.22C) RPMI culture medium (non-competitive) or (FIG. 22D) 100 nMunlabeled CD20 mAbs of the same clone (competitive).

FIG. 23 . Summarizing overview of molecular determinants of existing andnovel CD20 mAbs. Three distinct molecular determinants were described todetermine the MoA of CD20 mAbs: 1) epitope (positions of several CD20antibodies are indicated); 2) kinetics (k_(off) of the CD20 antibodieswere ranked from high (10⁻⁵) to low (10⁻⁶), and 3) elbow angle(commercially available CD20 mAbs (OFA, RTX and OBZ) were grouped inwide and narrow angle according to literature. The amino acid at Kabatposition 11 was described to influence the angle, and based on this, thenew chimeric mAbs were ordered according to the residue).

FIG. 24 . Amino acid sequence of suitable IgG1, IgA1 and IgA2 heavy andlight chain constant regions.

SEQ ID NO:3 Heavy chain IgG1 constant region CH1-3 and Hinge

SEQ ID NO:4 Heavy chain IgA1 constant region CH1-3 and Hinge

SEQ ID NO:5 Heavy chain IgA2 constant region CH1-3 and Hinge

SEQ ID NO-6 light chain constant region

FIG. 25 . Internalization of CD20 from B cells at 1 ug of antibody. Thenumber in the UMAB . . . reference in the figures refer to the variabledomains of antibody m . . . with the respective numbers and the constantregion as indicated.

FIGS. 26.1, 26.2, and 26.3 . Titration range of antibodies in CD20internalization of B cells.

FIGS. 27A, 27B. Analysis of in vitro efficacy of new chimeric IgA-CD20mAbs. (FIG. 27A) ADCC of IgA1-CD20 antibodies: Specific lysis of Daudicells in a chromium release assay with PMNs as effector cells(E:T=40:1). (FIG. 27B) ADCC of IgA2-CD20 antibodies: Specific lysis ofDaudi cells in a chromium release assay with PMNs as effector cells(E:T=40:1).

FIGS. 28A-28C. In vivo efficacy of IgA-CD20 mAbs. C57BL/6 mice (6mice/group) were injected intraperitoneally with 5×10e5 CellTraceVioletlabeled EL4-CD20 cells 16 hours prior to mAb (10 μg) or PBS treatment.The anti-tumor response was evaluated 24 hours later by determining theamount of remaining tumor cells in the peritoneal lavage with TruCounttubes (median±interquartile range)

FIG. 29 . Loss of CD20 expression occurs after IgG treatment, but notafter IgA treatment in vivo. C57BL/6 mice (6 mice/group) were injectedintraperitoneally with 5×10e5 CellTraceViolet labeled EL4-CD20 cells 16hours prior to mAb (10 μg) or PBS treatment. Subsequently, CD20expression was determined on these cells by flow cytometric analysis

FIG. 30 . CD19/CD24+ events with antibodies having a variable domain ofthe indicated antibody and the indicated constant regions, IgG1, IgA1 orIgA2.

DETAILED DESCRIPTION

The disclosure is concerned with antibodies that bind CD20. The CD20protein is also known under various other names such as MembraneSpanning 4-Domains A1; MS4A1; Membrane-Spanning 4-Domains Subfamily AMember 1; Leukocyte Surface Antigen Leu-16; CD20 Antigen; Bp35;B-Lymphocyte Cell-Surface Antigen B1; B-Lymphocyte Surface Antigen B1;CD20 Receptor, LEU-16; CVID5; MS4A2; B1; and S7. External Ids for MS4A1are HGNC: 7315; Entrez Gene: 931; Ensembl: ENSG00000156738; OMIM: 112210and UniProtKB: P11836.

Some of the names may or may not have also been used to refer to otherproteins than CD20. The names and sequence identifiers are given forreference purposes only. An antibody of the disclosure hinds to CD20 asexpressed on Ramos cells but also to other CD20 molecules as long as theepitope to which the antibody binds is available. Thus splicing variantsor mutant CD20 molecules (if any) will also be bound by an antibody ofthe disclosure as long as the epitope is available. The fact that theantibody binds to CD20 means that the antibody can bind to CD20 and doesnot imply that the antibody is actually bound to C D20. It also does notmean that the antibody does not bind to other proteins. Suchcross-reactivity is at present not known for an antibody of thisdisclosure, however, it is not expressly excluded that suchcross-reactivity may exist.

An antibody (Ab), also known as an immunoglobulin (Ig), is a large,typically Y-shaped protein. An antibody interacts with variouscomponents of the immune system. Some of the interactions are mediatedby its Fc region (located at the base of the “Y”), which containssite(s) involved in these interactions.

Antibodies are proteins belonging to the immunoglobulin superfamily.They typically have two heavy chains and two light chains. There areseveral different types of antibody heavy chains that define the fivedifferent types of crystallisable fragments (Fc) that may be attached tothe antigen-binding fragments. The five different types of Fc regionsallow antibodies to be grouped into five isotypes. An Fc region of aparticular antibody isotype is able to bind to its specific Fc receptor(FcR) thus allowing the antigen-antibody complex to mediate differentroles depending on which FcR it binds. The ability of an IgG antibody tobind to its corresponding FcR is modulated by the presence/absence ofinteraction sites and the structure of the glycan(s) (if any) present atsites within its Fc region. The ability of antibodies to bind to FcRshelps to direct the appropriate immune response for each different typeof foreign object they encounter.

Though the general structure of all antibodies is similar, a region atthe tip of the protein is extremely variable, allowing millions ofantibodies with slightly different tip structures, or antigen-bindingsites, to exist. This region is known as the hypervariable region. Theenormous diversity of antigen binding by antibodies is largely definedby the hypervariable region and the variable domain containing thehypervariable region.

An antibody of the disclosure is typically a full-length antibody. Theterm “full length antibody” is defined as comprising an essentiallycomplete immunoglobulin molecule, which however does not necessarilyhave all functions of an intact immunoglobulin. For the avoidance ofdoubt, a full length antibody has two heavy and two light chains. Eachchain contains constant (C) and variable (V) regions. A heavy chain of afull length antibody typically comprises a CH1, a CH2, a CH3, a VHregion and a hinge region. A light chain of a full length antibodytypically comprises a CL region and a VL region.

An antibody binds to antigen via the variable region domains containedin the Fab portion. An antibody variable domain comprises a heavy chainvariable region and a light chain variable region. Full lengthantibodies according to the disclosure encompass heavy and light chainswherein mutations may be present that provide desired characteristics.Full length antibodies should not have deletions of substantial portionsof any of the regions. However, IgG molecules wherein one or severalamino acid residues are substituted, inserted, deleted or a combinationthereof, without essentially altering the antigen bindingcharacteristics of the resulting antibody, are embraced within the term“full length” antibody. For instance, a “full length” antibody can havea substitution, insertion, deletion or a combination thereof, of between1 and 10 (inclusive) amino acid residues, preferably in non-CDR regions,wherein the deleted amino acids are not essential for the bindingspecificity of the antibody. [00%] The epitope that is recognized by anantibody of the disclosure, and/or minor contributing amino acidstherein were determined by, among others, positional amino acid scanwherein the amino acid was replaced by every other natural amino acid ina peptide containing the epitope and by a mutant screen of the CD20protein expressed on cells. The contribution of an amino acid to thebinding of an antibody to an epitope is preferably determined bycomparing the binding to a peptide comprising the epitope as such andthe same peptide but with an alanine at the position of the analyzedamino acid. An amino acid is relevant to the binding of the antibody tothe protein when a replacement with an alanine in the protein results ina decrease of binding of the antibody to 0-70% relative to theunmodified protein. This is also referred to a reduction of binding. Adecrease to 0-20% if the binding relative to the unmodified protein isregarded as loss of binding and a decrease to 21-70% relative to theunmodified protein is regarded as intermediate binding. The thusidentified amino acids are considered to be a major contributor or anintermediate contributor to the binding of the antibody to the protein.A binding of 71-100% was regarded as full binding. The amino acidconcerned is not regarded to contribute significantly to the binding ofthe antibody to the protein.

One of the antibodies provided by the disclosure is an antibody (A)comprising a mouse IgG2; a human IgG1, IgA1 or IgA2 constant region anda variable domain that can bind the epitope “EPANpSEK” (SEQ ID NO:31) onhuman CD20 expressed on Ramos cells and which antibody has an increasedPCD functionality when compared to Rituximab with a constant region ofthe same isotype. The antibody preferably further comprises a comparableor an increased CDC functionality when compared to Rituximab with aconstant region of the same isotype. Preferably the ADCC functionalityof the antibody is comparable or reduced when compared to Rituximab witha constant region of the same isotype. The epitope on CD20 that is boundby the antibody is “EPANpSEK” (SEQ ID NO:31). A capital letter, smallcase letter and bold indicates the relevance of the amino acid forbinding of the antibody to the peptide. A bold letter indicates that theamino acid is a major contributor to the binding of the antibody; asmall case letter indicates that the amino acid has an intermediatecontribution to the binding and a capital letter in plain text indicatesthat the amino acid has a small or not detectable contribution to thebinding of the antibody to the peptide. The antibody binds 20% or lessto a CD20 protein wherein one or more of the amino acids N or S in“EPANpSEK” (SEQ ID NO:31) have been replaced by an alanine, where thebinding is compared to the binding of the antibody an unmodified CD20protein.

Also provided is an antibody (A1) that can bind to an extracellular partof human CD20 expressed on Ramos cells comprising a variable domain witha heavy chain variable region and a light chain variable region, whereinthe heavy chain variable region comprises a CDR3 region with thesequence SNSYGSTYWYFDV (SEQ ID NO:21). The antibody (A1) has anincreased PCD functionality when compared to Rituximab with a constantregion of the same isotype. The antibody preferably further comprises acomparable or an increased CDC functionality when compared to Rituximabwith a constant region of the same isotype. Preferably the ADCCfunctionality of the antibody is comparable or reduced when compared toRituximab with a constant region of the same isotype. The heavy chainvariable region preferably comprises a CDR1, CDR2 and CDR3 region withthe sequence SYNLH (SEQ ID NO:26), AIYPGNGDTSYNQKFKG (SEQ ID NO:27) andSNSYGSTYWYFDV (SEQ ID NO:21), respectively. Preferably the heavy chainvariable region comprises the sequence of SEQ ID NO:1, with 0-5 aminoacid insertions, deletions, substitutions, additions or a combinationthereof at one or more positions other than positions of the amino acidsthat constitute the CDR1, CDR2 and CDR3 regions, wherein SEQ ID NO:1 hasthe sequence

QAYLQ QSGAE LVRPG ASVKM SCKAS GYTFT SYNLH WVKQTPRQGL EWIGA IYPGN GDTSY NQKFK GKATL TVDKS SSTAYMQLSR LTSED SAVYF CARSN SYGST YWYFD VWGTG TTVTV SS.

The light chain variable region of the antibody (A1) preferablycomprises the sequence of SEQ ID NO:2, with 0-5 amino acid insertions,deletions, substitutions, additions or a combination thereof at one ormore positions other than positions of the amino acids that constitutethe CDR1, CDR2 and CDR3 regions, wherein SEQ ID NO:2 has the sequence

QIVLS QSPAV LFASP GEKVT MTCRA RSSVS YMDWY QQKPRSSPKP WIYAT SNLAS GVPAR FSGSG SGTSY SLTIS RVEAEDAATY YCQQW TSNPP TFGSG TKLEI KRADA APTVS IFPPS S.

The antibody A1 preferably comprises a mouse IgG2; a human IgG1, IgG2,IgG3, IgG4, IgM, IgE, IgA heavy chain constant region or a combinationthereof. Preferably it comprises a human IgG1, IgG2, IgA1 or IgA2 heavychain constant region or a combination thereof. Preferably it comprisesa human IgG1 constant region. In a preferred embodiment, the heavy chainconstant region is a human IgA1 or human IgA2 heavy chain constantregion, preferably a human IgA2; more preferably a human IgA2m,preferably an IgA2 m1 or IgA2m2, preferably IgA2 m1 heavy chain constantregion. In another preferred embodiment, the antibody comprises a murineIgG2 region, preferably a IgG2c constant region.

In a preferred embodiment, the antibody A is an A1 antibody.

The antibody A or A1 preferably comprises a heavy chain and a lightchain wherein the heavy chain comprises the sequence of SEQ ID NO:1 andthe sequence of SEQ ID NO:3, 4 or 5 with 0-15 amino acid insertions,deletions, substitutions, additions or a combination thereof at one ormore positions other than positions of the amino acids that constitutethe CDR1, CDR2 and CDR3 regions.

The antibody A or A1 preferably comprises alight chain comprising thesequence of SEQ ID N0:2 and the sequence of SEQ ID NO:6 with 0-15 aminoacid insertions, deletions, substitutions, additions or a combinationthereof at one or more positions other than positions of the amino acidsthat constitute the CDR1, CDR2 and CDR3 regions.

The disclosure also provides an antibody (D) comprising a mouse IgG2; ahuman IgG1, IgA1 or IgA2 constant region and a variable domain that canbind the epitope “EPANPsEK” (SEQ ID NO:31) on human CD20 expressed onRamos cells and which antibody has an increased ADCC functionality whencompared to Rituximab a constant region of the same isotype. The epitopein CD20 that is bound by the antibody is “EPANPsEK” (SEQ ID NO:31). Acapital letter, small case letter and bold indicates the relevance ofthe amino acid for binding of the antibody to the peptide. A bold letterindicates that the amino acid is a major contributor to the binding ofthe antibody; a small case letter indicates that the amino acid has anintermediate contribution to the binding and a capital letter in plaintext indicates that the amino acid has a small or not detectablecontribution to the binding of the antibody to the peptide. The antibodybinds 20% or less to a CD20 protein wherein the amino acid N in“EPANpSEK” (SEQ ID NO:31) has been replaced by an alanine, whereby thebinding is compared to the binding of the antibody an unmodified CD20protein.

Also provided is an antibody (B1) that can bind to an extracellular partof human CD20 expressed on Ramos cells, the antibody comprising avariable domain with a heavy chain variable region and a light chainvariable region, wherein the heavy chain variable region comprises aCDR3 region with the sequence YYYGSSYGAMDY (SEQ ID NO:22). The antibodyB1 has an increased ADCC functionality when compared to Rituximab withthe same isotype constant region. The heavy chain variable regionpreferably comprises a CDR1, CDR2 and CDR3 region with the sequenceSYNMH (SEQ ID NO:28), GIYPGNGDTSYNQKFKG (SEQ ID NO:29) and YYYGSSYGAMDY(SEQ ID NO:22), respectively.

Preferably, the heavy chain variable region comprises the sequence ofSEQ ID NO:7, with 0-5 amino acid insertions, deletions, substitutions,additions or a combination thereof at one or more positions other thanpositions of the amino acids that constitute the CDR1, CDR2 and CDR3regions, wherein SEQ ID NO:7 has the sequence

QAYLQ QSGAE LVRPG ASVKM SCKAS GYTFT SYNMH WVKQTPRQGL EWIGG IYPGN GDTSY NQKFK GKATL TVDKS SSTAYMQLSS LTSED SAVYF CARYY YGSSY GAMDY WGQGT SVTVS S.

The light chain variable region of the antibody B1 preferably comprisesthe sequence of SEQ ID NO:8, with 0-5 amino acid insertions, deletions,substitutions, additions or a combination thereof at one or morepositions other than positions of the amino acids that constitute theCDR1, CDR2 and CDR3 regions, wherein SEQ ID NO:8 has the sequence

QIVLS QSPAI LSASP GEKVT MTCRA SSSVS YMHWY QQKPGSSPKP WIYAT SNLAS GVPAR FSGSG SGTSY SLTIS RVEAADAATY YCHQW TFNPP TFGGG TKLEI KRADA APTVS IFPPS S.

The antibody B1 preferably comprises a mouse IgG2; a human IgG1, IgG2,IgG3, IgG4, IgM, IgF, IgA heavy chain constant region or a combinationthereof. Preferably, it comprises a mouse IgG2; a human IgG1, IgG2, IgA1or IgA2 heavy chain constant region or a combination thereof. Preferablyit comprises a human IgG1 constant region. In a preferred embodiment,the heavy chain constant region is a human IgA1 or human IgA2 heavychain constant region, preferably a human IgA2; more preferably a humanIgA2m, preferably an IgA2m1 or IgA2m2, preferably IgA2 m1 heavy chainconstant region. In another preferred embodiment, the antibody comprisesa murine IgG2 region, preferably a IgG2c constant region.

In a preferred embodiment, the antibody B is a B1 antibody.

The antibody B or B1 preferably comprises a heavy chain and a lightchain wherein the heavy chain comprises the sequence of SEQ ID NO:7 andthe sequence of SEQ ID NO:3, 4 or 5 with 0-15 amino acid insertions,deletions, substitutions, additions or a combination thereof at one ormore positions other than positions of the amino acids that constitutethe CDR1, CDR2 and CDR3 regions.

The antibody B or B1 preferably comprises a light chain comprising thesequence of SEQ ID NO:8 and the sequence of SEQ ID NO:6 with 0-15 aminoacid insertions, deletions, substitutions, additions or a combinationthereof at one or more positions other than positions of the amino acidsthat constitute the CDR1, CDR2 and CDR3 regions.

The disclosure also provides an antibody (C) comprising a mouse IgG2; ahuman IgG1, IgA1 or IgA2 constant region and a variable domain that canbind the epitope “EPANpsEK” (SEQ ID NO:31) on human CD20 expressed onRamos cells and which antibody has an increased CDC functionality whencompared to Rituximab with a constant region of the same isotype.Preferably it comprises similar ADCC functionality as Rituximab with aconstant region of the same isotype. The epitope in CD20 that is boundby the antibody is “EPANpsEK” (SEQ ID NO:31). A capital letter, smallcase letter and bold indicates the relevance of the amino acid forbinding of the antibody to the peptide. A bold letter indicates that theamino acid is a major contributor to the binding of the antibody; asmall case letter indicates that the amino acid has an intermediatecontribution to the binding and a capital letter in plain text indicatesthat the amino acid has a small or not detectable contribution to thebinding of the antibody to the peptide. The antibody binds 20/or less toa CD20 protein wherein the amino acid N in “EPANpSEK” (SEQ ID NO:31) hasbeen replaced by an alanine, whereby the binding is compared to thebinding of the antibody an unmodified CD20 protein.

Also provided is an antibody (C1) that can bind to an extracellular partof human CD20 expressed on Ramos cells comprising a variable domain witha heavy chain variable region and a light chain variable region, whereinthe heavy chain variable region comprises a CDR3 region with thesequence TYYYGSSPYWSFDV (SEQ ID NO:23). The antibody has an increasedCDC functionality when compared to Rituximab with a constant region ofthe same isotype. Preferably it comprises a similar ADCC functionalityas Rituximab with a constant region of the same isotype. The heavy chainvariable region preferably comprises a CDR1, CDR2 and CDR3 region withthe sequence SYNMH (SEQ ID NO:28), AIYPGNGDTSYNQKFKG (SEQ ID NO:27) andTYYYGSSPYWSFDV (SEQ ID NO:23), respectively.

Preferably the heavy chain variable region comprises the sequence of SEQID NO:9, with 0-5 amino acid insertions, deletions, substitutions,additions or a combination thereof at one or more positions other thanpositions of the amino acids that constitute the CDR1, CDR2 and CDR3regions, wherein SEQ ID NO:9 has the sequence

QAYLQ QSGAE LVRPG ASVKM SCKAS GYTFA SYNMH WIKQTPRQGL EIWAA IYPGN GDTSY NQKFK GKATL TVDKS SSTAYMQLSS LTSED SAVYF CARTY YYGSS PYWSF DVWGT GTTVT VSS.

The light chain variable region of the antibody C1 preferably comprisesthe sequence of SEQ ID NO:10, with 0-5 amino acid insertions, deletions,substitutions, additions or a combination thereof at one or morepositions other than positions of the amino acids that constitute theCDR1, CDR2 and CDR3 regions, wherein SEQ ID NO:10 has the sequence

DIQMT QSPAS LSASV GETVT VTCGA SYNIY GALNW YQRKQGKSPQ LLIYG ATNLA DGMSS RFSGS GSGRQ YSLKI SSLHPDDVAT YYCQN VLSNP PTFGG GTKLE IKRAD AAPTV SIFPP SS.

The antibody C1 preferably comprises a mouse IgG2; a human IgG1, IgG2,IgG3, IgG4, IgM, IgE, IgA heavy chain constant region or a combinationthereof. Preferably, it comprises a human IgG1, IgG2, IgA1 or IgA2 heavychain constant region or a combination thereof. Preferably it comprisesa human IgG1 constant region. In a preferred embodiment, the heavy chainconstant region is a human IgA 1 or human IgA2 heavy chain constantregion, preferably a human IgA2; more preferably a human IgA2m,preferably an IgA2m1 or IgA2m2, preferably IgA2 m1 heavy chain constantregion. In another preferred embodiment, the antibody comprises a murineIgG2 region, preferably a IgG2b constant region.

In a preferred embodiment, the antibody C is a C1 antibody.

The antibody C or C1 preferably comprises a heavy chain and a lightchain wherein the heavy chain comprises the sequence of SEQ ID NO:9 andthe sequence of SEQ ID NO:3, 4, or 5 with 0-15 amino acid insertions,deletions, substitutions, additions or a combination thereof at one ormore positions other than positions of the amino acids that constitutethe CDR1, CDR2 and CDR3 regions.

The antibody C or C1 preferably comprises alight chain comprising thesequence of SEQ ID NO:10 and the sequence of SEQ ID NO:6 with 0-15 aminoacid insertions, deletions, substitutions, additions or a combinationthereof at one or more positions other than positions of the amino acidsthat constitute the CDR1, CDR2 and CDR3 regions.

Also provided is an antibody D1 that can bind to an extracellular partof human CD20 expressed on Ramos cells comprising a variable domain witha heavy chain variable region and a light chain variable region, whereinthe heavy chain variable region comprises a CDR3 region with thesequence SRLFDSSYGWYFDV (SEQ ID NO:24). The antibody has an increasedCDC and/or increased ADCC functionality when compared to Rituximab witha constant region of the same isotype. Preferably it comprises animproved ADCC functionality as Rituximab with a constant region of thesame isotype. The heavy chain variable region preferably comprises aCDR1, CDR2 and CDR3 region with the sequence SYNMH (SEQ ID NO:28),AIYPGNGDTSYNQKFKG (SEQ ID NO:27) and SRLFDSSYGWYFDV (SEQ ID NO:24),respectively.

Preferably the heavy chain variable region comprises the sequence of SEQID NO:11, with 0-5 amino acid insertions, deletions, substitutions,additions or a combination thereof at one or more positions other thanpositions of the amino acids that constitute the CDR1, CDR2 and CDR3regions, wherein SEQ ID NO:11 has the sequence

QAYLQ QSGAE LVRPG ASVKM SCKAS GYTFP SYNMH WVKQTPRQGL EWIGA IYPGN GDTSY NQKFK GKASQ TVDKS SSTVYMQLSS LTSAD SAVYF CARSR LFDSS YGWYF DVWGT GTTVT VSS.

The light chain variable region of the antibody D1 preferably comprisesthe sequence of SEQ ID NO:12, with 0-5 amino acid insertions, deletions,substitutions, additions or a combination thereof at one or morepositions other than positions of the amino acids that constitute theCDR 1, CDR2 and CDR3 regions, wherein SEQ ID NO:12 has the sequence

QIVLS QSPAI LSAYP GEKVT MTCRA RSSVS YIDWY QQKAGSSPKP WIYAT SNLAS GVPAR FSGSG SGTSY SLTIS RVEAEDAATY YCQQW TSNPP TFGGG TKLEI KRADA APTVS IFPPS S.

The antibody D1 preferably comprises a mouse IgG2; a human IgG1, IgG2,IgG3, IgG4, IgM, IgE, IgA heavy chain constant region or a combinationthereof. Preferably, it comprises a human IgG1, IgG2, IgA1 or IgA2 heavychain constant region or a combination thereof. Preferably it comprisesa human IgG1 constant region. In a preferred embodiment, the heavy chainconstant region is a human IgA1 or human IgA2 heavy chain constantregion, preferably a human IgA2; more preferably a human IgA2m,preferably an IgA2 m1 or IgA2m2, preferably IgA2 m1 heavy chain constantregion. In another preferred embodiment, the antibody comprises a murineIgG2 region, preferably a IgG2c constant region.

In a preferred embodiment, the antibody D is a D1 antibody.

The antibody D or D1 preferably comprises a heavy chain and a lightchain wherein the heavy chain comprises the sequence of SEQ ID NO:11 andthe sequence of SEQ ID NO:3, 4, or 5 with 0-15 amino acid insertions,deletions, substitutions, additions or a combination thereof at one ormore positions other than positions of the amino acids that constitutethe CDR1, CDR2 and CDR3 regions.

The antibody D or D1 preferably comprises a light chain with thesequence of SEQ ID NO:12 and the sequence of SEQ ID NO:6 with 0-15 aminoacid insertions, deletions, substitutions, additions or a combinationthereof at one or more positions other than positions of the amino acidsthat constitute the CDR1, CDR2 and CDR3 regions.

The disclosure also provides an antibody (E) comprising a mouse IgG2; ahuman IgG1, IgA1 or IgA2 constant region and a variable domain that canbind the epitope “EPANpSEK” (SEQ ID NO:31) on human CD20 expressed onRamos cells and which antibody has an increased CDC and/or increasedADCC functionality when compared to Rituximab with a constant region ofthe same isotype. The epitope in CD20 that is bound by the antibody is“EPANpSEK” (SEQ ID NO:31). A capital letter, small case letter and boldindicates the relevance of the amino acid for binding of the antibody tothe peptide. A bold letter indicates that the amino acid is a majorcontributor to the binding of the antibody; a small case letterindicates that the amino acid has an intermediate contribution to thebinding and a capital letter in plain text indicates that the amino acidhas a small or not detectable contribution to the binding of theantibody to the peptide. The antibody binds 20% or less to a CD20protein wherein one or more of the amino acids N or S in “EPANpSEK” (SEQID NO:31) have been replaced by an alanine, where the binding iscompared to the binding of the antibody an unmodified CD20 protein.

Also provided is an antibody (E1) that can bind to an extracellular partof human CD20 expressed on Ramos cells comprising a variable domain witha heavy chain variable region and a light chain variable region, whereinthe heavy chain variable region comprises a CDR3 region with thesequence SAYYGSNVWFFDV (SEQ ID NO:25). The antibody has an increased CDCand/or increased ADCC functionality when compared to Rituximab with aconstant region of the same isotype. The heavy chain variable regionpreferably comprises a CDR1, CDR2 and CDR3 region with the sequenceSYNLH (SEQ ID NO:26), AIYPGNGDTSYNQKFKG (SEQ ID NO:27) and SAYYGSNVWFFDV(SEQ ID NO:25), respectively.

Preferably the heavy chain variable region comprises the sequence of SEQID NO:13, with 0-5 amino acid insertions, deletions, substitutions,additions or a combination thereof at one or more positions other thanpositions of the amino acids that constitute the CDR1, CDR2 and CDR3regions, wherein SEQ ID NO:13 has the sequence

QAYLQ QSGAD LVRPG ASVKM SCKAS GFTFP SYNLH WVKQTPRQGL EWIGA IYPGN GDTSY NQKFK GKATL TVDKS SSTAYMQLSS LTSED SAVYF CARSA YYGSN VWFFD VWGTG TTVTV SS.

The light chain variable region of antibody E1 preferably comprises thesequence of SEQ ID NO:14, with 0-5 amino acid insertions, deletions,substitutions, additions or a combination thereof at one or morepositions other than positions of the amino acids that constitute theCDR1, CDR2 and CDR3 regions, wherein SEQ ID NO:14 has the sequence

QIVLS QSPAI LSASP GEKVT MICRA SSSVS YMDWY QQKPGSSPKP WIYAT SNLAS GVPTR FSGSG SGTSY SLTIS RVEAEDAATY YCQQW ISNPP TFGAG TKLDL KRADA APTVS IFPPS S.

The antibody E1 preferably comprises a mouse IgG2; a human IgG1, IgG2,IgG3, IgG4, IgM, IgE, IgA heavy chain constant region or a combinationthereof. Preferably, it comprises a human IgG1, IgG2, IgA1 or IgA2 heavychain constant region or a combination thereof. Preferably it comprisesa human IgG1 constant region. In a preferred embodiment, the heavy chainconstant region is a human IgA1 or human IgA2 heavy chain constantregion, preferably a human IgA2; more preferably a human IgA2m,preferably an IgA2 m1 or IgA2m2, preferably IgA2 m1 heavy chain constantregion. In another preferred embodiment, the antibody comprises a murineIgG2 region, preferably an IgG2c constant region.

In a preferred embodiment, the antibody E is an E1 antibody.

The antibody E or E1 preferably comprises a heavy chain and a lightchain wherein the heavy chain comprises the sequence of SEQ ID NO:13 andthe sequence of SEQ ID NO:3, 4, or 5 with 0-15 amino acid insertions,deletions, substitutions, additions or a combination thereof at one ormore positions other than positions of the amino acids that constitutethe CDR1, CDR2 and CDR3 regions.

The antibody E or E1 preferably comprises a light chain with thesequence of SEQ ID NO:14 and the sequence of SEQ ID NO:6 with 0-15 aminoacid insertions, deletions, substitutions, additions or a combinationthereof at one or more positions other than positions of the amino acidsthat constitute the CDR1, CDR2 and CDR3 regions.

The functionality of an antibody can be compared to Rituximab with aconstant region of the same isotype. This is preferably the sameconstant region. Some amino acid differences may be present in theconstant regions, such amino acid differences can, for instance, beintroduced by somatic cell hypermutation. Between 0-5 amino aciddifferences are typically allowed, although more is also possible. For acomparison of functionality it is preferred that the constant regions ofantibody and rituximab are the same.

An antibody of the disclosure can have a heavy chain variable regionwith 0-5 amino acid insertions, deletions, substitutions, additions or acombination thereof at one or more positions with respect to thesequence indicated by the respective SEQ ID NO, wherein the one or morepositions are not positions in the CDR1, CDR2 and CDR3 regions. Thesequence of the CDRs is thus as indicated in the respective SEQ ID NO.It is preferred that the heavy chain variable region has 0-4 amino acidinsertions, deletions, substitutions, additions or a combination thereofat one or more positions with respect to the sequence indicated by therespective SEQ ID NO, wherein the one or more positions are notpositions in the CDR1, CDR2 and CDR3 regions. It is preferred that theheavy chain variable region has 0-3, more preferably 0-2, morepreferably 0-1 amino acid insertions, deletions, substitutions,additions or a combination thereof at one or more positions with respectto the sequence indicated by the respective SEQ ID NO, wherein the oneor more positions are not positions in the CDR1, CDR2 and CDR3 regions.In a preferred embodiment, a heavy chain variable region in the antibodyof the disclosure has 0 amino acid insertions, deletions, substitutions,additions or a combination thereof with respect to the sequence of theSEQ ID NO indicated.

An antibody of the disclosure can have a light chain variable regionwith 0-5 amino acid insertions, deletions, substitutions, additions or acombination thereof at one or more positions with respect to thesequence indicated by the respective SEQ ID NO, wherein the one or morepositions are not positions in the CDR1, CDR2 and CDR3 regions. Thesequence of the CDRs is thus as indicated in the respective SEQ ID NO.It is preferred that the light chain variable region has 0-4 amino acidinsertions, deletions, substitutions, additions or a combination thereofat one or more positions with respect to the sequence indicated by therespective SEQ ID NO, wherein the one or more positions are notpositions in the CDR1, CDR2 and CDR3 regions. It is preferred that thelight chain variable region has 0-3, more preferably 0-2, morepreferably 0-1 amino acid insertions, deletions, substitutions,additions or a combination thereof at one or more positions with respectto the sequence indicate by the respective SEQ ID NO, wherein the one ormore positions are not positions in the CDR1, CDR2 and CDR3 regions. Ina preferred embodiment, a light chain variable region in the antibody ofthe disclosure has 0 amino acid insertions, deletions, substitutions,additions or a combination thereof with respect to the sequence of theSEQ ID NO indicated.

A heavy chain of an antibody of the disclosure can have 0-15 amino acidinsertions, deletions, substitutions, additions or a combination thereofat one or more positions with respect to the sequence indicate by therespective SEQ ID numbers wherein the one or more positions are notpositions in the CDR1, CDR2 and CDR3 regions. The sequence of the CDRsis thus as indicated in the respective SEQ ID NO. It is preferred thatthe heavy chain has 0-10, amino acid insertions, deletions,substitutions, additions or a combination thereof at one or morepositions with respect to the sequence indicate by the respective SEQ IDnumbers, wherein the one or more positions are not positions in theCDR1, CDR2 and CDR3 regions. The heavy chain can have 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14 and 15, amino acid insertions, deletions,substitutions, additions or a combination thereof at one or morepositions with respect to the sequence indicate by the respective SEQ IDnumbers, wherein the one or more positions are not positions in theCDR1, CDR2 and CDR3 regions. It is preferred that the heavy chain has0-3, more preferably 0-2, more preferably 0-1 amino acid insertions,deletions, substitutions, additions or a combination thereof at one ormore positions with respect to the sequence indicate by the respectiveSEQ ID numbers, wherein the one or more positions are not positions inthe CDR1, CDR2 and CDR3 regions. In a preferred embodiment, the heavychain in the antibody of the disclosure has 0 amino acid insertions,deletions, substitutions, additions or a combination thereof withrespect to the sequence of the SEQ ID numbers indicated.

Alight chain of an antibody of the disclosure can have 0-15 amino acidinsertions, deletions, substitutions, additions or a combination thereofat one or more positions with respect to the sequence indicate by therespective SEQ ID numbers wherein the one or more positions are notpositions in the CDR1, CDR2 and CDR3 regions. The sequence of the CDRsis thus as indicated in the respective SEQ ID NO. It is preferred thatthe light chain has 0-10, amino acid insertions, deletions,substitutions, additions or a combination thereof at one or morepositions with respect to the sequence indicate by the respective SEQ IDnumbers, wherein the one or more positions are not positions in theCDR1, CDR2 and CDR3 regions. The light chain can have 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14 and 15, amino acid insertions, deletions,substitutions, additions or a combination thereof at one or morepositions with respect to the sequence indicate by the respective SEQ IDnumbers, wherein the one or more positions are not positions in theCDR1, CDR2 and CDR3 regions. It is preferred that the light chain has0-3, more preferably 0-2, more preferably 0-1 amino acid insertions,deletions, substitutions, additions or a combination thereof at one ormore positions with respect to the sequence indicate by the respectiveSEQ ID numbers, wherein the one or more positions are not positions inthe CDR1, CDR2 and CDR3 regions. In a preferred embodiment, the lightchain in the antibody of the disclosure has 0 amino acid insertions,deletions, substitutions, additions or a combination thereof withrespect to the sequence of the SEQ ID numbers indicated.

An antibody A, B, C, D, E, A1, B1, C1, D1, or E1 as described herein canbe a bispecific antibody comprising one variable domain that binds anantigen other than the indicated epitope on an extracellular part ofCD20 as expressed on Ramos cells. The other antigen is preferably CD19,CD64, CD32, CD16, CD3 and CD47. In a preferred embodiment, an antibodyA, B, C, D, E, A1, B1, C1, D1, or E1 comprises two variable domains thateach bind the same epitope on an extracellular part of CD20 as expressedon Ramos cells, wherein the epitope is as indicated for the variabledomain of antibody A, B, C, D, E, A1, B1, C1, D1, or E1. The antibody A,B, C, D, E, A1, B1, C1, D1, or E1 preferably comprises two identicalvariable domains. The antibody A, B, C, D, E, A1, B1, C1, D1, or E1preferably comprises two variable domains that each bind the sameepitope and comprise the same VH and the same VL sequence.

The disclosure further provides a CAR-T receptor comprising a variabledomain of an antibody A, B, C, D, E, A1, B1, C1, D1, or E1 as describedherein. The variable domain comprises a heavy chain variable region anda variable light chain region of the respective antibodies, each with0-5 amino acid insertions, deletions, substitutions, additions or acombination thereof at one or more positions other than positions of theamino acids that constitute the CDR1, CDR2 and CDR3 regions. Thesequence of the respective heavy and light chain variable regions isindicated in FIG. 18 .

For instance, a CAR-T receptor preferably comprises a variable domain ofantibody A1. In a preferred embodiment, the variable domain comprisesthe amino acid sequence of the heavy and light chain variable regions ofSEQ ID NOS:1 and 2, each with 0-5 amino acid insertions, deletions,substitutions, additions or a combination thereof at one or morepositions other than positions of the amino acids that constitute theCDR1, CDR2 and CDR3 regions.

A CAR-T receptor preferably comprises a variable domain of antibody B1.In a preferred embodiment, the variable domain comprises the amino acidsequence of the heavy and light chain variable regions of SEQ ID NOS:7and 8, each with 0-5 amino acid insertions, deletions, substitutions,additions or a combination thereof at one or more positions other thanpositions of the amino acids that constitute the CDR1, CDR2 and CDR3regions.

A CAR-T receptor preferably comprises a variable domain of antibody C1.In a preferred embodiment, the variable domain comprises the amino acidsequence of the heavy and light chain variable regions of SEQ ID NOS:9and 10, each with 0-5 amino acid insertions, deletions, substitutions,additions or a combination thereof at one or more positions other thanpositions of the amino acids that constitute the CDR1, CDR2 and CDR3regions.

A CAR-T receptor preferably comprises a variable domain of antibody D1.In a preferred embodiment, the variable domain comprises the amino acidsequence of the heavy and light chain variable regions of SEQ ID NOS:11and 12, each with 0-5 amino acid insertions, deletions, substitutions,additions or a combination thereof at one or more positions other thanpositions of the amino acids that constitute the CDR1, CDR2 and CDR3regions.

A CAR-T receptor preferably comprises a variable domain of antibody E1.In a preferred embodiment, the variable domain comprise the amino acidsequence of the heavy and light chain variable regions of SEQ ID NOS:13and 14, each with 0-5 amino acid insertions, deletions, substitutions,additions or a combination thereof at one or more positions other thanpositions of the amino acids that constitute the CDR1, CDR2 and CDR3regions.

Also provided is a T-cell comprising a CAR-T cell receptor of thedisclosure for use in adoptive cell transfer. The use is preferably forthe treatment of an individual that has a CD20 positive neoplasm such asa CD20 positive B-cell lymphoma; hairy cell leukemia; B-cell chroniclymphocytic leukemia, or melanoma comprising administering to theindividual in need thereof an antibody as described herein. It is alsouseful in the treatment of children with B-cell malignancies andpediatric leukemia patients that have a B-cell disease after stem celltransplantation.

A reason for changing an amino acid at a certain position can beimmunogenicity.

Other reasons include but are not limited to improving production orhomogeneity of the antibody. Antibodies of the present disclosure havevariable heavy and variable light chain regions derived from a murinebackground. Antibodies with such variable domains can be used in humans.Presently it is preferred to de-immunize such variable domains.De-immunization typically involves the modification of the murinesequence into a more human sequence whenever possible. Typically suchmodifications are directed toward removing one or more T-cell epitopesor one more B-cell epitopes from the variable domain. In a preferredembodiment, the disclosure provides an antibody A, B, C, D, E, A1, B1,C1, D1, or E1 wherein one or more (human) T-cell epitopes have beenremoved by replacement of at least one amino acid of the epitope with adifferent amino acid. Often it is sufficient to substitute the so-called“anchor” amino acid. Suitable replacement amino acids can be obtainedfrom somatic cell hypermutants of the particular VH or VL. Replacementwith an amino acid that is naturally present at that position in a humanantibody is preferred. In a preferred embodiment, the disclosureprovides an antibody A, B, C, D, E, A1, B1, C1, D1, or E1 wherein one ormore (human) B-cell epitopes have been removed by replacement of atleast one amino acid of the epitope with a different amino acid. Oftenit is sufficient to substitute only one amino acid of the epitope.Suitable replacement amino acids can be obtained from somatic cellhypermutants of the particular VH or VL. Replacement with an amino acidthat is naturally present at that position in a human antibody ispreferred. Preferably a variable domain of the disclosure is modifiedwith respect to one or more exterior residues. Such residues are readilyencountered by the immune system and are preferably selectively replacedwith human residues to provide a hybrid molecule that comprises either aweakly immunogenic or substantially non-immunogenic surface. Suitablereplacement amino acids can be obtained from somatic cell hypermutantsof the particular VH or VL. Replacement with an amino acid that isnaturally present at that position in a human antibody is preferred. Thedisclosure thus further provides an antibody A, B, C, D, E, A1, R1, C1,D1, or E1 that comprises a humanized heavy chain variable region, ahumanized light chain variable region or a combination thereof.

The disclosure further provides an antibody A, B, C, D, E, A1, B1, C1,D1, or E1 for use in the treatment of a disease in an individual. Alsoprovided is an antibody A, B, C, D, E, A1, B1, C1, D1, or E1 for use inthe treatment of a disease that involves too many B cells, overactive Bcells, and/or dysfunctional B cells. Further provided is antibody A, B,C, D, E, A1, B1, C1, D1, or E1 for use in the treatment of a CD20positive neoplasm such as a CD20 positive B-cell lymphoma; hairy cellleukemia; B-cell chronic lymphocytic leukemia, and melanoma.

Also provided is the use of an antibody A, B, C, D, E, A1, B1, C1, D1,or E1 for the manufacture of a medicament for the treatment of a diseasethat involves too many B cells, overactive B cells, and/or dysfunctionalB cells. Also provided is the use of an antibody A, B, C, D, E, A1, B1,C1, D1, or E1 for the manufacture of a medicament for the treatment of aCD20 positive neoplasm such as a CD20 positive B-cell lymphoma; hairycell leukemia; B-cell chronic lymphocytic leukemia, and melanoma.

Also provided is a method for the treatment of an individual that has adisease that involves too many B cells, overactive B cells, and/ordysfunctional B cells comprising administering to the individual in needthereof an antibody A, B, C, D, E, A1, B1, C1, D1, or E1.

Further provided is a method for the treatment of an individual that hasa CD20 positive neoplasm such as a CD20 positive B-cell lymphoma; hairycell leukemia; B-cell chronic lymphocytic leukemia, or melanomacomprising administering to the individual in need thereof an antibodyA, B, C, D, E, A1, B1, C1, D1, or E1.

The disclosure also provides a nucleic acid molecule that codes for aheavy chain, a light chain and/or a variable region thereof. Such anucleic acid molecule is typically but not exclusively a ribonucleicacid (RNA) or a deoxyribonucleic acid (DNA). Alternative nucleic acidsare available for a person skilled in the art, such as, for instance,peptide nucleic acids (PNA).

Examples of nucleic acid molecules provided by the disclosure are:

-   -   a nucleic acid molecule that codes for a heavy chain or light        chain of an antibody as described herein;    -   a nucleic acid molecule that codes for the CDR3 of a heavy or        light chains of an antibody as described herein;    -   a nucleic acid molecule that codes for the CDR1, CDR2 and CDR3        of a heavy or light chain of an antibody as described herein;        and    -   a nucleic acid molecule that codes for the variable region of a        heavy chain or of a light chain of an antibody as described        herein.    -   a nucleic acid molecule that codes for a heavy chain variable        region comprising a CDR3 region with the sequence SNSYGSTYWYFDV.    -   a nucleic acid molecule that codes for a heavy chain variable        region comprising CDR1, CDR2 and CDR3 region with the sequence        SYNLH(SEQ ID NO:26) AIYPGNGDTSYNQKFKG(SEQ ID NO:27) and        SNSYGSTYWYFDV (SEQ ID NO:21), respectively.    -   a nucleic acid molecule that codes for a heavy chain variable        region with the sequence of SEQ ID NO:1, with 0-5 amino acid        insertions, deletions, substitutions, additions or a combination        thereof at one or more positions other than positions of the        amino acids that constitute the CDR1, CDR2 and CDR3 regions,        wherein SEQ ID NO:1 has the sequence QAYLQ QSGAE LVRPG ASVKM        SCKAS GYTFT SYNLH WVKQT PRQGL EWIGA IYPGN GDTSY NQKFK GKATL        TVDKS SSTAY MQLSR LTSED SAVYF CARSN SYGST YWYFD VWGTG TTVTV SS.    -   a nucleic acid molecule that codes for alight chain variable        region with the sequence of SEQ ID NO:2, with 0-5 amino acid        insertions, deletions, substitutions, additions or a combination        thereof at one or more positions other than positions of the        amino acids that constitute the CDR1, CDR2 and CDR3 regions,        wherein SEQ ID NO:2 has the sequence QIVLS QSPAV LFASP GEKVT        MTCRA RSSVS YMDWY QQKPR SSPKP WIYAT SNLAS GVPAR FSGSG SGTSY        SLTIS RVEAE DAATY YCQQW TSNPP TFGSG TKLEI KRADA APTVS IFPPS S.    -   a nucleic acid molecule that codes for a heavy chain that        comprises the sequence of SEQ ID NO:1 and the sequence of SEQ ID        NO:3, SEQ ID NO:4, or SEQ ID NO:5 with 0-15 amino acid        insertions, deletions, substitutions, additions or a combination        thereof at one or more positions other than positions of the        amino acids that constitute the CDR1, CDR2 and CDR3 regions.    -   a nucleic acid molecule that codes for a light chain that        comprises the sequence of SEQ ID NO-2 and the sequence of SEQ ID        NO:6 with 0-15 amino acid insertions, deletions, substitutions,        additions or a combination thereof at one or more positions        other than positions of the amino acids that constitute the        CDR1, CDR2 and CDR3 regions.    -   a nucleic acid molecule that codes for a heavy chain variable        region with a CDR3 region with the sequence YYYGSSYGAMDY (SEQ ID        NO:22).    -   a nucleic acid molecule that codes for a heavy chain variable        region that comprises a CDR1, CDR2 and CDR3 region with the        sequence SYNMH (SEQ ID NO:28), GIYPGNGDTSYNQKFKG (SEQ ID NO:29)        and YYYGSSYGAMDY (SEQ ID NO:22), respectively.    -   a nucleic acid molecule that codes for a heavy chain variable        region comprises the sequence of SEQ ID NO:7, with 0-5 amino        acid insertions, deletions, substitutions, additions or a        combination thereof at one or more positions other than        positions of the amino acids that constitute the CDR1, CDR2 and        CDR3 regions, wherein SEQ ID NO:7 has the sequence QAYLQ QSGAE        LVRPG ASVKM SCKAS GYTFT SYNMH WVKQT PRQGL EWIGG IYPGN GDTSY        NQKFK GKATL TVDKS SSTAY MQLSS LTSED SAVYF CARYY YGSSY GAMDY        WGQGT SVTVS S.    -   a nucleic acid molecule that codes for alight chain variable        region that comprises the sequence of SEQ ID NO:8, with 0-5        amino acid insertions, deletions, substitutions, additions or a        combination thereof at one or more positions other than        positions of the amino acids that constitute the CDR1, CDR2 and        CDR3 regions, wherein SEQ ID NO:8 has the sequence QIVLS QSPAI        LSASP GEKVT MTCRA SSSVS YMHWY QQKPG SSPKP WIYAT SNLAS GVPAR        FSGSG SGTSY SLTIS RVEAA DAATY YCHQW TFNPP TFGGG TKLEI KRADA        APTVS IFPPS S.    -   a nucleic acid molecule that codes for a heavy chain that        comprises the sequence of SEQ ID NO:7 and the sequence of SEQ ID        NO:3, SEQ ID NO:4, or SEQ ID NO:5 with 0-15 amino acid        insertions, deletions, substitutions, additions or a combination        thereof at one or more positions other than positions of the        amino acids that constitute the CDR 1, CDR2 and CDR3 regions.    -   a nucleic acid molecule that codes for a light chain comprising        the sequence of SEQ ID NO:8 and the sequence of SEQ ID NO:6 with        0-15 amino acid insertions, deletions, substitutions, additions        or a combination thereof at one or more positions other than        positions of the amino acids that constitute the CDR1, CDR2 and        CDR3 regions.    -   a nucleic acid molecule that codes for a heavy chain variable        region that comprises a CDR3 region with the sequence        TYYYGSSPYWSFDV (SEQ ID NO:23).    -   a nucleic acid molecule that codes for a heavy chain variable        region that comprises a CDR1, CDR2 and CDR3 region with the        sequence SYNMH (SEQ ID NO:28), AIYPGNGDTSYNQKFKG (SEQ ID NO:27)        and TYYYGSSPYWSFDV (SEQ ID NO:23), respectively.    -   a nucleic acid molecule that codes for a heavy chain variable        region comprises the sequence of SEQ ID NO:9, with 0-5 amino        acid insertions, deletions, substitutions, additions or a        combination thereof at one or more positions other than        positions of the amino acids that constitute the CDR1, CDR2 and        CDR3 regions, wherein SEQ ID NO:9 has the sequence QAYLQ QSGAE        LVRPG ASVKM SCKAS GYTFA SYNMH WIKQT PRQGL EWIAA IYPGN GDTSY        NQKFK GKATL TVDKS SSTAY MQLSS LTSED SAVYF CARTY YYGSS PYWSF        DVWGT GTTVT VSS.    -   a nucleic acid molecule that codes for alight chain variable        region that comprises the sequence of SEQ ID NO:10, with 0-5        amino acid insertions, deletions, substitutions, additions or a        combination thereof at one or more positions other than        positions of the amino acids that constitute the CDR1, CDR2 and        CDR3 regions, wherein SEQ ID NO:10 has the sequence DIQMT QSPAS        LSASV GETVT VTCGA SYNIY GALNW YQRKQ GKSPQ LLIYG ATNLA DGMSS        RFSGS GSGRQ YSLKI SSLHP DDVAT YYCQN VLSNP PTFGG GTKLE IKRAD        AAPTV SIFPP SS.    -   a nucleic acid molecule that codes for a heavy chain that        comprises the sequence of SEQ ID NO:9 and the sequence of SEQ ID        NO:3, 4, or 5 with 0-15 amino acid insertions, deletions,        substitutions, additions or a combination thereof at one or more        positions other than positions of the amino acids that        constitute the CDR1, CDR2 and CDR3 regions.    -   a nucleic acid molecule that codes for a light chain comprising        the sequence of SEQ ID NO:10 and the sequence of SEQ ID NO:6        with 0-15 amino acid insertions, deletions, substitutions,        additions or a combination thereof at one or more positions        other than positions of the amino acids that constitute the        CDR1, CDR2 and CDR3 regions.    -   a nucleic acid molecule that codes for heavy chain variable        region that comprises a CDR3 region with the sequence        SRLFDSSYGWYFDV (SEQ ID NO:24).    -   a nucleic acid molecule that codes for a heavy chain variable        region that comprises a CDR1, CDR2 and CDR3 region with the        sequence SYNMH (SEQ ID NO:28), AIYPGNGDTSYNQKFKG (SEQ ID NO:27)        and SRLFDSSYGWYFDV (SEQ ID NO:24), respectively.    -   a nucleic acid molecule that codes for a heavy chain variable        region that comprises the sequence of SEQ ID NO:11, with 0-5        amino acid insertions, deletions, substitutions, additions or a        combination thereof at one or more positions other than        positions of the amino acids that constitute the CDR1, CDR2 and        CDR3 regions, wherein SEQ ID NO:11 has the sequence QAYLQ QSGAE        LVRPG ASVKM SCKAS GYTFP SYNMH WVKQT PRQGL EWIGA IYPGN GDTSY        NQKFK GKASQ TVDKS SSTVY MQLSS LTSAD SAVYF CARSR LFDSS YGWYF        DVWGT GTTVT VSS.    -   a nucleic acid molecule that codes for alight chain variable        region that comprises the sequence of SEQ ID NO:12, with 0-5        amino acid insertions, deletions, substitutions, additions or a        combination thereof at one or more positions other than        positions of the amino acids that constitute the CDR1, CDR2 and        CDR3 regions, wherein SEQ ID NO:12 has the sequence QIVLS QSPAI        LSAYP GEKVT MTCRA RSSVS YIDWY QQKAG SSPKP WIYAT SNLAS GVPAR        FSGSG SGTSY SLTIS RVEAE DAATY YCQQW TSNPP T1FGGG TKLEI KRADA        APTVS IFPPS S.    -   a nucleic acid molecule that codes for a heavy chain that        comprises the sequence of SEQ ID NO:11 and the sequence of SEQ        ID NO:3, 4, or 5 with 0-15 amino acid insertions, deletions,        substitutions, additions or a combination thereof at one or more        positions other than positions of the amino acids that        constitute the CDR1, CDR2 and CDR3 regions.    -   a nucleic acid molecule that codes for a light chain with the        sequence of SEQ ID NO:12 and the sequence of SEQ TD NO:6 with        0-15 amino acid insertions, deletions, substitutions, additions        or a combination thereof at one or more positions other than        positions of the amino acids that constitute the CDR1, CDR2 and        CDR3 regions.    -   a nucleic acid molecule that codes for a heavy chain variable        region that comprises a CDR3 region with the sequence        SAYYGSNVWFFDV (SEQ ID NO:25).    -   a nucleic acid molecule that codes for a heavy chain variable        region that comprises a CDR1, CDR2 and CDR3 region with the        sequence SYNLH (SEQ ID NO:26), AIYPGNGDTSYNQKFKG (SEQ ID NO:27)        and SAYYGSNVWFFDV (SEQ ID NO:25), respectively.    -   a nucleic acid molecule that codes for a heavy chain variable        region that comprises the sequence of SEQ ID NO:13, with 0-5        amino acid insertions, deletions, substitutions, additions or a        combination thereof at one or more positions other than        positions of the amino acids that constitute the CDR1, CDR2 and        CDR3 regions, wherein SEQ ID NO:13 has the sequence QAYLQ QSGAD        LVRPG ASVKM SCKAS GFTFP SYNLH WVKQT PRQGL EWIGA IYPGN GDTSY        NQKFK GKATL TVDKS SSTAY MQLSS LTSED SAVYF CARSA YYGSN VWFFD        VWGTG TTVTV SS.    -   a nucleic acid molecule that codes for a light chain variable        region that comprises the sequence of SEQ ID NO:14, with 0-5        amino acid insertions, deletions, substitutions, additions or a        combination thereof at one or more positions other than        positions of the amino acids that constitute the CDR1, CDR2 and        CDR3 regions, wherein SEQ ID NO:14 has the sequence QIVLS QSPAI        LSASP GEKVT MTCRA SSSVS YMDWY QQKPG SSPKP WIYAT SNLAS GVPTR        FSGSG SGTSY SLTIS RVEAE DAATY YCQQW ISNPP TFGAG TKLDL KRADA        APTVS IFPPS S.    -   a nucleic acid molecule that codes for a heavy chain that        comprises the sequence of SEQ ID NO:13 and the sequence of SEQ        ID NO:3, 4, or 5 with 0-15 amino acid insertions, deletions,        substitutions, additions or a combination thereof at one or more        positions other than positions of the amino acids that        constitute the CDR1, CDR2 and CDR3 regions.    -   a nucleic acid molecule that codes for a light chain with the        sequence of SEQ ID NO:14 and the sequence of SEQ ID NO:6 with        0-15 amino acid insertions, deletions, substitutions, additions        or a combination thereof at one or more positions other than        positions of the amino acids that constitute the CDR1, CDR2 and        CDR3 regions.

A nucleic acid molecule as described herein is preferably used for theproduction of an antibody A, B, C, D, E, A1, B1, C1, D1 or E1 asdescribed herein by a cell comprising the nucleic acid molecule. Anucleic acid molecule comprising two or more of the indicated sequencescomprises such sequence in the order and linkage suitable for theproduction of an antibody A, B, C, D, E, A1, B1, C1, D1 or E1 asdescribed herein by a cell comprising the nucleic acid molecule. Thenucleic acid molecule preferably further comprises one or more sequencesfor the expression of an antibody as described. Non-limiting examples ofsuch sequences are a promoter, a termination sequence, an enhancer, anintron etc. Such sequences are not necessarily present on the nucleicacid molecule as such sequences can be provided in cis by theintegration site of the nucleic acid molecule in, for instance, achromosome of a cell, or a vector comprising the nucleic acid molecule.Suitable integration sites in a cellular chromosome can easily bedetermined and targeted, for instance, by means of homologousrecombination.

Further provided is a cell that comprises one or more nucleic acidmolecules as described herein.

Further provided are means and methods for the production of an antibodyas described herein using a nucleic acid molecule of the disclosure or acell comprising a nucleic acid molecule of the disclosure.

A nucleic acid according to the disclosure, is, for instance, comprisedin a cell. When the nucleic acid is expressed in the cell thetranslation product of the nucleic acid molecule can be incorporatedinto an antibody of the disclosure. The disclosure thus also provides acell comprising a nucleic acid molecule according to the disclosure. Thedisclosure further provides a cell comprising a nucleic acid molecule ofthe disclosure and that is capable of producing an antibody of thedisclosure. Further provided is a method for producing an antibody ofthe disclosure comprising culturing a cell comprising expressing one ormore nucleic acid molecules that code for an antibody of the disclosureand harvesting the antibody from the culture medium, the cell or acombination thereof. The cell is preferably an animal cell, morepreferably a mammalian cell. The cell is preferably a cell that isnormally used for the production of an antibody for use in humans.Non-limiting examples of such cells are CHO, NSO and PER.C6 cells. Cellsmay specifically designed to suit certain purposes, for instance, mostcell lines used for the production of antibodies have been adapted forgrowth in suspension, in high densities and other properties. For thepurpose of the disclosure a suitable cell is any cell capable ofcomprising and preferably of producing an antibody according to thedisclosure.

PCD function of an antibody as claimed is preferably measured in amethod based on annexin V positivity in flow cytometry (as described,for instance, in Example 3).

ADCC function of an antibody as claimed is preferably measured in amethod based on B cell depletion in blood drawn from healthy volunteersmeasured in flow cytometry by the decrease of CD24 positive cells,negative for myeloid markers, or a classical chromium release assay (asdescribed, for instance, in Example 1).

CDC of an antibody as claimed is preferably measured in a method basedon 7AAD positivity in flow cytometry (see, for instance, Example 1).

EXAMPLES Example 1. Novel CD20 antibodies and molecular determinantsthat govern functional properties of these antibodies Materials andMethods

Antibodies

CD20 mAbs were generated by cellular immunization and standard fusion ofspleen cells to obtain hybridomas. Isotypes were determined by FACS(rat-anti-mIgG2a-biotin (cross-reactive with mlgG2c; BD),goat-anti-mIgG2b-Fcγ-RPE (SouthernBiotech), goat-anti-mIgG3-PerCP(Jackson)). mAbs were purified after growth in serum free medium byaffinity chromatography using HiTRap rProteinA FF columns (GEHealthcare). Bound protein was eluted with 0.1 M sodium acetate pH 2.5(Sigma Aldrich) and directly neutralized with 1 M Tris-HCl pH 8.8.Chimeric IgG1 (IgG1) mAbs were generated by cloning the variable regions(synthesized by Shinegene) into Lonza expression vectors(pEE14.4-kappaLC, pEE14.4-IgG1. The L11V mutation was introduced bysite-directed mutagenesis. All IgG1 mAbs were produced by transienttransfection of HEK293F cells⁽²²⁾ and purified by ProteinA affinitychromatography. All mAbs were dialyzed to PBS (Sigma-Aldrich) and theconcentration was determined using the following formula: OD value at280 nm/correction factor (mIgG 1.36; chIgG 1.35).

RTX (chIgG1; Pharmacy UMC Utrecht), OFA (human (h)IgG1; Pharmacy UMCUtrecht), mouse (m)IgG2a-CD20-7D8 (m7D8) and mIgG2a-CD20-11B8 (m11B8)(kindly provided by Genmab B V, Utrecht, The Netherlands), B1 (mIgG2a,kindly provided by Mark Cragg, Southhampton, UK) and mIgG2a-RTX (mRTX)(Invivogen) were dialyzed to PBS if needed. Abs were labeled with FTTC(Thermo Scientific) or Alexa647 (Molecular Probes) following themanufacturer's instructions.

Cell lines

SKBR3 cells (ATCC) were retrovirally transduced with human CD20 togenerate SKBR3-CD20 cells. A subclone stably expressing CD20 wasestablished by limiting dilution. EL4-CD20 cells were generated aspreviously described.⁽²³⁾ Daudi, Ramos, Raji (ATCC), and the abovementioned cells were cultured in culture medium containingRPMI-1640+HEPES+glutamine (Invitrogen) supplemented with 10% fetal calfserum (FCS), 100 U/mL penicillin and 100 μg/mL streptomycin (LifeTechnologies) at 37° C./5% CO₂. FreeStyle™ HEK293F cells (Invitrogen)were cultured in FreeStyle™ 293 expression medium (Invitrogen) at 37°C./8% CO₂ on an orbital shaker.

Determination of Consensus Amino Acid Sequence of Variable Regions

From frozen dry cell pellets, RNA was isolated and purified using aRNeasy mini kit (Qiagen). Purified RNA (2 μg) was used as templates forcDNA synthesis with a RevertAid H Minus First Strand cDNA synthesis kit(Fermentas). Next, variable light (VL) and variable heavy (VH) regionswere amplified in PCR reactions (see Supplementary Materials andMethods). Gel-purified PCR products were cloned into the pCRII bluntTOPO vector (Life Technologies) and transformed into DH5a E. coli.Plasmid DNA was isolated from several clones using a QiAprep spinminiprep kit (Qiagen). Subsequently, consensus DNA sequences of VL andVH regions from each mIgG-CD20 mAb were determined by DNA sequencing(Macrogen) of several plasmid clones containing insert. Based on thesequence information of several clones of each mAb, the consensusV-region sequences were determined and subsequently the amino acidsequences were deduced.

Homotypic Aggregation and Cell Viability Assay

0.4×10⁵ EL4-CD20 cells together with 1 μg/mL Ab in culture medium wereplated out in a 96-well plate, and incubated for 24 hours at 37° C./5%CO₂. In the cross-linking conditions the following Ahs were added 30minutes later: 20 μg/mL rabbit-F(ab′)2-anti-hIgG (Jackson) or 50 μg/mLrabbit-F(ab′)2-anti-mIgG (Jackson). Homotypic aggregation was assessedsemi-quantitatively using an EVOS microscope (20× magnification). Cellviability was determined by AnnexinV-PE and 7-AAD (Pharmingen) staining,following the manufacturer's instructions

Human PBMC ADCC

ADCC assays with ⁵¹Cr-labeled target cells were performed as previouslydescribed.^(22,24) Briefly, PBMC isolated from healthy individuals(MiniDonorDienst UMC Utrecht) by Ficoll separation (GE Healthcare) werecombined with ⁵¹Cr-labeled Daudi cells (effector-to-target ratio=100:1)and CD20 mAb in dilution. After 4 hours incubation at 37° C./5% CO₂, thesupernatant was harvested and counted in a liquid scintillation counter(MicroBeta; Perkin Elmer). Lysis was calculated using the followingformula: % lysis=((counts of sample−minimum release)/(maximumrelease−minimum release))×100. Daudi cells with PBMC in culture mediumor in medium supplemented with 2.5% Triton X-100 (Roche Diagnostics)were used to determine minimum and maximum release, respectively.

CDC Assay

10⁵ cells (Daudi, Ramos, Raji) were plated out in a 96-well plate andpre-incubated for 30 minutes at room temperature with Ab diluted inculture medium. Human serum (pooled from 8 healthy donors) as complementsource (final concentration 15.5%) was added and the preparation keptfor 15 minutes at 37° C./5% CO₂. The degree of complement-mediated lysiswas assessed by staining with 7-AAD (Pharming). Cells were measured on aFACS Canto II(BD).

CD20 Peptide Library Design and Screen

The design of the CD20 peptide library was described elsewhere.⁽²⁰⁾Briefly, both linear and cyclic peptides, with loops of different sizes,covering the 2 extracellular loops were directly synthesized ontopolypropylene plates.^((25,26)) Linear and discontinuous epitopes werereconstructed and for each peptide and a full amino acid scan wasincluded (see Supplementary Materials and Methods).

Recognition of peptides by the CD20 mAb was assessed in a PepScan-basedELISA as previously described.⁽²⁰⁾ Briefly, 1 μg/mL mAb was added toplates with covalently linked peptides and incubated overnight at 4° C.After extensive washing, bound mAb was detected with a HRP-linkedrabbit-anti-mIgG (DakoCytomation) for 1 hour at 25° C. Unbound detectionAb was washed away. Plates were developed using ABTS substrate and colordevelopment quantified using a CCD camera and an image-processingsystem.

Mutant Screen

HEK293F cells were transfected with vectors coding for wild-type ormutated CD20 using 293fectin (Invitrogen) following the manufacturer'sinstructions. One day post-transfection, cells were harvested, washed inFACS buffer and stained for 30 minutes with 5 μg/mL CD20 mAbs on ice.mAb binding was detected with a goat-anti-hIgG-APC or goat-anti-mIgG-APCAb (Jackson). Cells were measured on a FACS Canto II (BD).

Ligand Tracer

Kinetics analysis of interactions between the mAbs and CD20 wasperformed by using the LigandTracer green Technology (RidgeviewInstruments AB). 106 SKBR3-CD20 cells were seeded on one side of a 10 cmculture dish (Greiner) as target cell area and association of 10 nMFITC-labeled CD20 mAb was measured. For non-competitive or competitivedissociation, labeled mAb solution was replaced by RPMI culture mediumor 100 nM unlabeled mAb of the same clone, respectively. Analysis wasperformed using an OneToOne fitting model in TraceDrawer (RidgeviewInstruments AB).

EL4-CD20 Lymphoma Model

C57BL/6 mice were purchased from Janvier (France) or bred in thefacilities. 4-6 mice/group were injected intraperitoneally (i.p.) with5×10⁵ CellTraceViolet (10 μM, Invitrogen)-labeled EL4-CD20 cells. After16 hours, mice were treated with mAb or PBS (100 μL) injected i.p. Aperitoneal lavage with PBS containing 5 mM EDTA was performed after 24hours and the amount of remaining tumor cells was determined usingTruCount tubes (BD). All experiments were approved by the Animal EthicalCommittee of the UMC Utrecht.

Supplementary Materials and Methods

Amplification of VH and VL Regions

The PCR reaction contained 25 ng cDNA, 1× AccuPrime mix (LifeTechnologies), 25 pmol 5′ and 3′ primers (Biolegio) annealing to thecDNA encoding the signal peptide and to the constant region of theantibody, respectively, and AccuPrime™ Pfx DNA Polymerase (LifeTechnologies) according to manufacturer's instructions. Amplificationconsisted of initialization at 95° C. for 2 minutes, followed by 34cycles of denaturation at 95° C. for 30 seconds, annealing at 55 or 60°C. for 30 seconds, and extension at 68° C. for 2.5 minutes, finally the34 cycles were followed by an extension step at 68° C. for 7 minutes.PCR tubes were maintained at 4° C. until further processing.

Binding Assay

10⁵ Daudi cells were plated out in a 96-well plate and incubated withmIgG CD20 mAbs (in PBS) for 45 minutes on ice. After washing, bound CD20mAbs were detected with goat-F(ab′)2-anti-mIgG(H+L)-APC (SouthernBiotech) and subsequently measured on a FACS Canto II (BD).

Detailed Description of Peptide Library Used in Epitope MappingExperiment

Single-domain and double-domain peptides covering the 2 extracellularCD20 loops were used to reconstruct linear and discontinuous epitopes.For each peptide a full positional aa scan (i.e., each position isreplaced by all other aa) was included. The second part of the libraryconsisted of cyclic peptides by introducing cysteine-based loops usingthe Pepscan technology (www.pepscan.n1). The following peptides wereincluded: (a) all overlapping linear 34-mers covering the large loop,including a full alanine-scan of each 34-mer peptide; (b) 34 overlappingpeptides from the large loop and 1 peptide from the small loop werecombined in a matrix of 35×35 with each other in order to mapdiscontinuous epitopes that are spread over two different parts; (c) alloverlapping linear 15-mers covering complete CD20; (d) a full positionalaa scan (excluding cysteine) of YNCEPANPSEKNSPSTQYCYS (SEQ ID NO:16);(e) one double-looped small loop covering peptide; (f) single-loopedpeptides of varying size covering the large loop to captureconformational epitopes that depend on a particular loop size and (g)all overlapping single-looped 15-mers covering the completeextracellular sequence of the CD20 molecule.

FACS-Based Dissociation

10⁵Daudi cells were incubated with 10 μg/mL Alexa647-labeled mAb for 1hour at 37° C. and then pelleted and resuspended in either 100 μg/mLunlabeled mAb or complete RPMI culture medium. Cells were left at 37°C./5% CO₂ and measured on a FACS Canto H at indicated time points todetermine the level of cell-bound mAbs over time. The remaining meanfluorescence intensity (MFI) at each time point was expressed as apercentage of the initial MFI.

Results

New CD20 mAbs Exhibit Type I Characteristics

A panel of novel CD20 mAbs was generated and characterized in detail.After fusion of spleen B-cells of 6 immunized mice, 17 stable hybridomaclones producing mIgG2c—, mIgG2b- and mIgG3-CD20 mAbs (Table 1) wereobtained. For 14 out of 17 CD20 mAbs, unique VH and VL chain sequencepairs with differing degrees of relatedness (FIGS. 1.1 and 1.2 ) wereretrieved. Comparison of the new mIgG-CD20 mAbs with available Type I/IICD20 mAbs revealed that the CDR3 of the VH chain is the least conserved(27-63% identity for new mIgG-CD20 mAbs compared to RTX). For furtherstudies, 11 CD20 mAbs were purified. Their binding was comparable toType I CD20 mAbs m7D8 (derived from same panel as OFA with comparableproperties¹⁴ but expressed as mIgG2a mAb) and mRTX (FIG. 19 ).

Next, their MoA was determined. Neither PCD (FIG. 2A) nor homotypicaggregation (FIG. 20 ), both strongly elicited by Type II mAbs, wereinduced by the new CD20 mAbs in the absence of a cross-linking Ab.

As a shared feature of Type I/II mAbs, specific cell lysis in an ADCCassay using Daudi cells as targets and PMBC as effectors (FIG. 2B) wasdetermined. A comparable degree of lysis was obtained with allmIgG2c-CD20 mAbs. In contrast, m1gG2b mAbs were less effective.Remarkably, CDC activity, the MoA only elicited by Type I mAbs, differedconsiderably amongst the CD20 mAbs (FIG. 2C).

Variation of CDC Capacity of New mIgG2c-CD20 mAbs

For further comparisons, focus was placed on the mIgG2c mAbs as theyexhibited higher levels of effector functions. CDC was analyzed over awider concentration range on Daudi cells, a cell line sensitive forcomplement-mediated lysis due to high CD20 and low complement-regulatoryprotein (CRP) expression (FIG. 3A). m1 and m9 showed the highest CDCactivity, in particular, between 0.1-3 μg/mL mAb. m10 displayedintermediate CDC potency, and m2 was the least potent. The better CDCinduction by m1 compared to m2 was also detected in assays with Ramosand Raji cells, which have decreasing CD20 and increasing CRP expression(FIG. 3B).

New CD20 mAbs with Overlapping, but Distinct Epitopes

To study a possible correlation between functional properties and theepitope, m1 and m2, both mIgG2c-CD20 mAbs but highest/lowest in CDC,were subjected to a detailed epitope mapping analysis applying thePepScan-technology. Critical residues of m1 were identified to be¹⁶⁸EPANPSEK¹⁷⁵ by using linear (FIG. 21A) and circular (FIG. 4A, left)peptides with a positional amino acid scan covering the largerextracellular loop. In contrast, the signal for binding of m2 to thelinear (FIG. 21B) and circular (FIG. 4A, right) peptide was rather low.However, a signal decrease below the wild-type (WT) binding signal onlyoccurred within the ¹⁶⁸EPANPSEK¹⁷⁵ sequence motif. This suggests thatthe epitope of both mAbs is located on the larger loop in the sameregion, however their binding characteristics are different. The datasuggest that m1 binds to a linear epitope, whereas m2 might bind to aconformational epitope.

After identifying the epitope of two new CD20 mAbs based on peptidescanning, defining the recognition sites on properly folded CD20 for thecomplete mAb panel was of interest next. Therefore, a rough epitopemapping experiment with two CD20 mutants was first performed, with aminoacids mutated in either the small (T159K/N163D/N166D) or larger(A170S/P172S) loop of CD20 (FIG. 4B). None of the new CD20 mAbs showedan impaired binding upon mutations in the binding region of m7D8(T159K/N163D/N166D). Mutations in the RTX epitope region (A170S/P172S)resulted in diminished binding of all new CD20 mAbs to a differentextent (0-20% of WT binding. RTX, m1, m6, m7, m17; 20-30% of WT binding:m3, m4, m5, m10; 50-80% of WT binding: m2, m9, m11).

To determine crucial amino acids required for binding within the largerextracellular loop, a single mutant library spanning the¹⁶⁸PANPSEKNSP¹⁷⁸ sequence (FIG. 4C) was used. All 11 evaluated CD20 mAbswith unique sequence pairs showed distinct binding patterns. None of the11 new CD20 mAbs showed loss of binding upon mutations of N176 or E174,two residues found to impact binding of the Type II mAbs B1 and m11BR,respectively. The binding of m9 remained untouched by any mutant. Allother new CD20 mAbs showed an impaired binding to the CD20 molecule uponmutation of N171, as seen for mRTX. The two single mutants A170P andP172A made it possible to identify the contributing amino acids withinthe A170S/P172S double-mutant. The A170P mutation did not affect mAbbinding. In contrast, the P172A mutant influenced binding of all newCD20 mAbs, except for m2, m9 and m11. Residue S173 was identified to beimportant for the binding of mRTX, m1, m4 and m10. Overall, the epitopeof most new CD20 mAbs can be narrowed down to ¹⁷⁰ANPSE¹⁷⁴, howeverdifferent amino acids are required for binding.

CD20 mAbs Show Variation in Binding Stability

A slow Ab off-rate might favor the formation of Ab-C1q complexes, thefirst component of the classical complement pathway. FACS-baseddissociation assays hinted at unique dissociation behaviors of m1 and m2(FIGS. 22A, 22B), suggesting a faster off/on-rate for m2. Tocharacterize interaction kinetics and affinity of mAbs, the LigandTracertechnology was used to record real-time binding and dissociation of Absto cellular receptors. Kinetics analysis was performed for all 4mIgG2c-CD20 mAbs under non-competitive (FIG. 5A, left) and competitive(FIG. 5A, right) conditions and detected differences in their bindingbehavior. All mAbs had a similar apparent affinity in the low tosub-nanomolar range (Table 3). Binding rate constants were comparablefor all mAbs. The major difference in kinetics was observed in thedissociation phase. OFA, m9 and m10 dissociated very slowly, while m1,m2 and RTX had a more pronounced biphasic release pattern, meaning thatone fraction of the mAbs is dissociating rapidly while another fractionis more stable bound (determined by InteractionMap analysis; data notshown). Also, except for m9, dissociation under competitive conditionsis faster than under non-competitive conditions (FIG. 5C), indicatingthat dissociated mAbs rebind to the cell under non-competitiveconditions. This occurs, for example, when other receptors are in closeproximity or when Ab binding is stabilized by multiple binding, such asin Ab-(CD20)₂ or CD20-Ab-C1q complexes. m2 has the largest fraction offast releasing Abs and dissociation is strongest affected bycompetition. The formation of more stable complexes due to multipleinteractions is substantiated by the observation that competition has astronger impact on the release of labeled mAb when incubation times areprolonged (FIG. 22C, 22D).

Chimerization can Impact Functional Properties

During the humanization of B-Ly1 to OBZ, a leucine to valine mutation atKabat position 11 in the VH chain was introduced. The insertion of aV11L mutation into the OBZ sequence resulted in the loss of its PCDcapacity and widened its elbow angle. To evaluate whether this residuegenerally alters the MoA of CD20 mAbs, the reverse mutation (L11V) wasintroduced into the chimerized versions of m1 (IgG1-CD20-1) andm2(IgG1-CD20-2), respectively. Binding, ADCC and CDC were comparable ata saturating concentration (data not shown). Surprisingly, in a PCDassay with EL4-CD20 cells (FIG. 6 ) IgG1-CD20-1 induced PCD, a propertylost upon insertion of the V11L mutation. In contrast, for IgG1-CD20-2neither the parental nor the mutated versions induced PCD. Thisindicates that the MoA can change upon chimerization and that the elbowangle determining region plays a crucial role in it.

New CD20 mAbs Exhibit Variation in Anti-Tumor Response in Vivo

As mIgG2c-CD20 mAbs were the most potent in vitro, their in vivo potencyin a short low tumor burden model was studied, previously established inthe lab.⁽²⁷⁾ In this model CDC was shown to be the dominant effectormechanism for tumor cell eradication. Since m1 and m2 displayed thebiggest differences in vitro, these two mAbs were used first todetermine the effective Ab concentration (FIG. 7A). A clearconcentration-dependent anti-tumor response was observed, and at 1 μgmAb a complete clearance of the tumor cells was achieved with m1 but notm2. Testing the efficacy of the remaining mIgG2c mAbs at 1 μg, m9 andm10 show that they performed comparable to m1 (FIG. 7B).

Discussion

For the treatment of B-cell malignancies, RTX (chIgG1), OFA (hIgG1) andOBZ (glycoengineered hIgG1 Fc-region for enhanced ADCC) targeting CD20are FDA approved. In vitro they induce tumor cell killing mediated byeither ADCC and CDC (Type I; RTX and OFA) or ADCC and PCD (Type II;OBZ). It is likely that certain patient groups with e.g., lessbeneficial FcγR polymorphisms or different types of B-cell malignanciesmight benefit from treatments with highly complement active or apoptosisinducing mAbs. In order to develop more effective CD20 therapeutic mAbs,further understanding of the underlying characteristics dictating theMoA of CD20 mAbs is important. Generating Type-specific CD20 mAbsremains a challenge, not only due to the low innate immunogenicity ofthe CD20 molecule itself, but also due to the lack of understanding,which Ab properties are relevant for function. New CD20 mAbs were raisedusing an efficient in-house developed immunization method and purified11 mAbs for further characterization. Studying functional propertiesrevealed all new CD20 mAbs to exhibit Type I characteristics as theyinduce CDC, but not PCD. In the panel, mIgG2b was the most frequentisotype, followed by mIgG2c and mIgG3. This is indicative for morematured Abs, since mIgG2c and mIgG2b Abs are further advanced in theclass switching order of the heavy chain in C57BL/6 mice (5′cμ-cδ-cγ3-cγ1-cγ2b-cγ2c-cε-cα3′).⁽²⁸⁾ The diversity in the variableregions indicated that affinity-matured CD20 mAbs were raised.

To investigate underlying properties for the MoA of the new mAbs, theepitope was determined, binding kinetics and structural properties basedon a mutation at Kabat position 11. For the epitope, the C-terminal partof the larger loop of CD20 was found to be relevant for B1 and OBZbinding, suggesting that this epitope is important for the Abs' Type IIfeatures.^((18,29)) However, the similar location of the epitope of OBZand B1 is most likely an incomplete explanation as B-Ly1, the parentalAb of OBZ,⁽³⁰⁾ does not display the complete Type II characteristicsbefore humanization.⁽³¹⁾ Additionally, the epitope of Type II mAb 11B8is not overlapping, but comprised of amino acids located on the smalland large extracellular loop, similar to 7D8 and OFA. 10 out of 11 ofthe mAbs bound in the same region as RTX. The data suggest that residueS173 is relevant in determining the type of epitope. Binding of m1(linear epitope) is abrogated upon mutation of residue S173. Incontrast, m2 binding (conformational epitope), is much less affected.This varying recognition results in an alteration of the bindingbehavior and in depth analysis suggests that it has an impact on thecapacity of the mAb to create secondary interactions. Also the recentlysuggested V11L mutation in GA101, reversing the mAbs Type IIcharacteristics, is not a universal explanation. Sequence comparisonconfirmed that Type I CD20 mAbs, but also B1 and 11B8, all carry aleucine at this position. Interestingly, the chimerized mAbs derivedfrom m1 exhibited PCD capacity. Subsequent introduction of valine atKabat position 1 abrogated all PCD activity. This points at aninvolvement of the elbow-hinge angle, however it is inconclusive as towhich residues are important, as the amino acid sequences of the VHframework of m1 and m2 are identical. Kabat position 11 with B1 might beinvolved, as next to the elbow-hinge angle, the lack of binding to N171was implicated as a second requirement.⁽¹²⁾

Concerning the binding kinetics, previous findings on the contributionof the off-rate of CD20 mAbs to CDC were contradictory. Mitigating theoff-rate of RTX with a non-complement activating reagent increased itsCDC activity.⁽¹⁴⁾ Also veltuzumab, a humanized IgG1 mAb with CDRsidentical to RTX except for 1 residue, displays a slower off-rate thanRTX and higher CDC capacity.⁽³²⁾ However, no CDC enhancement was inducedby RTX mutants with a slower off-rate.⁽³³⁾ With the panel of mIgG2c-CD20mAbs, which varied in their ability to elicit CDC, no correlation wasfound between good CDC and slow dissociation as determined inLigandTracer experiments. Generally, functional characteristics couldnot be clearly correlated to the observed differences in the interactionkinetics or affinity and/or the epitope. Recently, it was suggested thatthe superior CDC activity of hIgG1 mAbs is due to Fc-Fc interactionsafter antigen-binding, resulting in the formation of hexamers, whicheventually facilitate C1q-binding.⁽³⁴⁾ Introduction of mutationsstrengthening the Fc-interactions in weak complement-activating Absresulted in enhanced CDC activity.³⁵

The binding kinetics analysis revealed, however, divergent dissociationpatterns and suggests that the CD20 mAbs differ in their ability to bindother molecules, leading to stabilization of Ab binding; whereas mostOFA and m10 rapidly form a stable interaction, for m1, m2, and RTX amore heterogeneous pattern was observed. The release of m2 was clearlyaffected by self-competition. These data point to multi-site binding ofthe mAbs, which differs among the panel. The fraction of mAbs, whichreleases more quickly reduces upon increasing the incubation time. Thesefindings raise the question of the time dependency and type of complexformation and its effect on lipid raft formation by Type I mAbs, andeventually the influence on CDC induction.

In vitro, the new mIgG2c-CD20 mAbs could be grouped in strong (m1 andm9), intermediate (m10) and weaker (m2) complement inducers. In vivo,m1, m9 and m10 performed comparable, whereas m2 was less effective. Thissuggests that differences detected in in vitro CDC assays were notnoticeable with the used model. This is likely a model dependent effect,and different when other in vivo models are used. Characteristics thatgovern the functional properties of CD20 mAbs are indicated in FIG. 23 .

Example 2 Material and Methods

Antibodies

Rituximab (RTX, chimeric (ch)IgG1), Ofatumumab (OFA, human (h)IgG1), andTrastuzumab (TRA, humanized IgG1) were obtained from the Pharmacy of theUMC Utrecht.

The generation of the new chimeric IgG1 mAbs IgG1-CD20-1 and IgG1-CD20-2was described in Chapter 7. IgG1-CD20-7, IgG1-CD20-9, and IgG1-CD20-10were made by U-Protein (Utrecht), who produced the mAbs in HEK293Tcells. All mAbs were dialyzed to 1× PBS (Sigma-Aldrich) and theconcentration was determined using the following formula:

$\frac{{OD}{value}{at}280{nm}}{{correction}{factor}\left( {{chlgG}1.35} \right)\text{?}}$?indicates text missing or illegible when filed

Cell Lines

Daudi cells (ATCC) were cultured in RPMI culture medium containingRPMI-1640+HEPES+glutamine (Invitrogen) supplemented with 10% fetal calfserum (FCS) and 100 U/mL penicillin and 100 μg/mL streptomycin (1× P/S;Life Technologies) at 37° C./5% CO₂. EL4-CD20 cells were generated aspreviously described⁽²³⁾ and cultured in RPMI culture medium.

Binding Assay

10⁵ Daudi cells were plated out in a 96-well plate and incubated withIgG1-CD20 mAbs (in PBS) for 45 minutes on ice. After washing, bound CD20mAbs were detected with goat-F(ab)₂-anti-hIgG-RPE (Southern Biotech) andsubsequently measured on a FACS Canto II (BD).

Human PBMC ADCC

ADCC assays with ⁵¹Cr-labeled target cells were performed as previouslydescribed.⁽²²⁾ PBMCs were isolated from healthy individuals(MiniDonorDienst UMC Utrecht) by Ficoll separation (GE Healthcare;Sigma-Aldrich). ¹⁵Cr-labeled Daudi cells were combined with PBMCs(effector-to-target ratio=50:1) and CD20 mAb in dilution. After 4 hoursincubation at 37° C./5% CO₂, the supernatant was harvested and countedin a liquid scintillation counter (MicroBeta; Perkin Elmer). Lysis wascalculated using the following formula:

${\%{lysis}} = {\frac{{counts}_{sample} - {counts}_{{minimal}{release}}}{{counts}_{{maximum}{release}} - {counts}_{{minimal}{release}}} \times 100.}$

Daudi cells with effector cells in RPMI culture medium or in mediumsupplemented with 2.5% Triton X-100 (Roche Diagnostics) were used todetermine minimal and maximum release, respectively.

CDC Assay

10⁵ cells Daudi cells were plated out in a 96-well plate andpre-incubated for 30 minutes at room temperature with mAbs diluted inRPMI culture medium. Human serum (pooled from 8 healthy donors) ascomplement source (final concentration 15.5%) was added and thepreparation kept for the 15 minutes at 37° C./5% CO₂. The degree ofcomplement-mediated lysis was assessed by staining dead cells with 7-AAD(BD Pharmingen). Cells were measured on a FACS Canto II (BD).

Cell Viability Assay

4×10⁴ EL4-CD20 cells together with 1 μg/mL Ab in RPMI culture mediumwere plated out in a 96-well plate, and incubated for 24 hours at 37°C./5% CO₂. In the cross-linking condition 20 μg/mLrabbit-F(ab′)2-anti-hIgG (Jackson ImmunoResearch) was added 30 minutesafter start of incubation. Cell viability was determined by AnnexinV-PEand 7-AAD (BD Pharmingen) staining, following the manufacturer'sinstructions.

B-Cell Depletion Assay

Blood from healthy donors was collected in Hirudin blood tubes andstored on ice until use. mAbs were diluted in RPMI-1640+HEPES+glutamine(Invitrogen) and unprocessed blood was added. After incubation of theplates for 60 minutes at 37° C./5% C02 samples were kept on ice for allfurther steps. Leukocytes and CD19⁺ cells were stained by addingmouse-anti-hCD45-PO (Life Technologies) and mouse-anti-hCD19-APC(Biolegend) for 30 minutes. Subsequent lysis of erythrocytes with 1× BDPharm Lyse Lysing buffer (BD) supplemented with 5 mM EDTA pH 8(Sigma-Aldrich), 7-AAD (BD Pharmingen) and Cyto/Cal Multifluor PlusViolet Flow Cytometer Alignment Beads (Thermo Scientific) was followedby measurement on a FACS Canto II (BD).

EL4-CD20 Lymphoma Model

C57BL/6 mice were purchased from Janvier (Le Genest Saint Isle, France).6 mice/group were injected intraperitoneally (i.p.) with 5×10⁵CellTraceViolet (10 μM, Invitrogen)-labeled EL4-CD20 cells. After 16hours, mice were treated with 10 μg mAb or PBS (100 μL) injected i.p. Aperitoneal lavage with 1× PBS containing 5 mM EDTA was performed after24 hours. The amount of remaining tumor cells was determined usingTruCount tubes (BD). All experiments were approved by the Animal EthicalCommittee of the UMC Utrecht.

Results

From the panel of previously characterized mIgG CD20 mAbs, 5 promisingcandidates for chimerization were selected. Four of these were mIgG2cmAbs (m1, m2, m9, and m10; see Example 1) which exhibited a diversefunctional pattern in vitro. Among the mIgG2b mAbs, m7 displayed thehighest complement-dependent cytotoxicity. After cloning the variableregions into human IgG1 constant region and human kappa light chain (LC)expression vectors, the mAbs in either HEK293F or HEK293T cells wereproduced. Subsequently, purified chIgG1 mAbs were subjected to apreliminary in vitro characterization.

First, the antigen-binding capacity of all chimerized CD20 mAbs weretested. They specifically bound to CD20 positive Daudi cells (FIG. 8 ).Their binding pattern was comparable to the commercially available CD20mAbs RTX (chIgG1) and OFA (hIgG1).

Next, their potential to eradicate tumor cells in two different assayswas assessed. First, tumor cell killing was measured in a classicalchromium-release ADCC assay, by incubating ⁵¹Cr-labeled Daudi cells withmAb and PBMCs (effector to target ratio=50:1) (FIG. 9A). With thechimeric IgG1-CD20 mAbs clearer differences in their ADCC capacity couldbe seen, compared to their mouse versions (Example 1). The percentage ofmaximal lysis at 1 μg/mL mAb ranged between 38% (IgG1-CD20-1) and 49%(IgG1-CD20-10). Though maximal lysis was slightly higher for thecommercial mAbs RTX and OFA, at lower concentrations, IgG1-CD20-2,IgG1-CD20-9, and IgG1-CD20-10 performed better than RTX.

Tumor cell killing by activation of the complement system is the secondeffector mechanism commonly induced by IgG1-CD20 mAbs. The chIgG1-CD20mAbs showed a concentration dependent tumor cell lysis (FIG. 9B). AllmAbs induced a higher degree of CDC compared to RTX. IgG1-CD20-2remained the least potent one. IgG1-CD20-1 and IgG1-CD2-10 performedsimilar, and IgG1-CD20-7 even better than OFA at lower concentrations.

The last mechanism of action described to be induced by CD20 mAbs, istumor cell death by programmed cell death (PCD). This property isstrongly induced by so called Type II CD20 mAbs like Obinutuzumab(OBZ)⁽¹²⁾ and B1 (mIgG2a CD20 mAb).⁽¹³⁾ RTX was shown to kill tumorcells to a lower extent via the same caspase-independent pathway.⁽¹³⁾None of the parental mouse CD20 mAbs induced neither homotypicaggregation, as sign for cell death induction, nor an increase in 7-AADand AnnexinV positivity. As described in Example 1, IgG1-CD20-1 inducedPCD of EL4-CD20 cells. Here, the full IgG1-CD20 mAb panel was compared,however, for the other 4 CD20 mAbs, chimerization did not change thesecharacteristics (FIG. 9C).

Finally, the degree of B-cell depletion in an autologous whole bloodassay was assessed. An increase of 7AAD⁺ CD19⁺ B cells upon incubationwith mAb at any concentration was not observed, the focus was only onthe number of viable CD19⁺ B cells (FIG. 10A). A mAb concentrationdependent decline of viable CD19⁺ B cells (FIG. 10B) was detected. RTXas the weakest CDC inducer performed the worst. No clear differencescould be observed between the new IgG1-CD20 mAbs.

Eventually, using the previously established EL4-CD20 low tumor burdenmodel, it was determined at a fully saturating concentration of 10 μgmAb the efficacy of the chimeric IgG1-CD20 mAbs. No major differenceswere detectable, but all mAbs showed a potent induction of tumor cellkilling in vivo (FIG. 11 ).

Discussion

Starting with mouse sequences, an important step before humanization isto test chimeric mAbs for their therapeutic potential. Five new chimericIgG1-CD20 mAbs were generated, and they all induced tumor cell killingin vitro and in vivo. Differences in their ADCC and CDC activityhowever, did not translate into a better anti-tumor response in vivo.

OBZ, a potent ADCC and PCD inducer, was shown to prolong survival ofSCID mice more efficiently than RTX in a NHL xenograft model.⁽¹²⁾However, the individual contribution of ADCC or PCD to the efficacy hasnot been evaluated yet.

The potential of CD20 mAbs has been studied extensively in whole bloodB-cell depletion assays. Commonly, 4 hours to 24 hours incubation timeswere used.^((12, 50, 51)) It was shown that OBZ exhibits superior andfaster killing of B cells in an autologous setting of healthy but alsopatient donor blood compared to RTX. However, in those assays alleffector mechanisms are likely to contribute. The mAbs was incubated for1 hour with unprocessed blood. This set-up is beneficial for theevaluation of complement-mediated rather than effector cell-mediatedtumor cell killing. CDC is quickly induced, whereas NK-cell-mediatedtumor cell eradication requires longer incubation times. This possiblyexplains the poor results obtained with RTX, the weakest complementactivator. The effectiveness of the new CD20 mAbs in the B-celldepletion assay correlated with obtained CDC results. Next to donorvariability, a decrease of CD19 expression within the lymphocyte gatewas detected, indicating an influence of CD20 mAb treatment on CD19. Itwas previously shown that RTX treatment induces CD19 shaving ortrogocytosis, a neutrophil- and monocyte/macrophage-mediatedmechanism.⁽⁹⁻¹¹⁾ As this seems to be a very fast effect, alternativestrategies to determine the B-cell depletion potential of CD20 mAbs inan autologous setting are being investigated. Here, CD24 as a secondaryB-cell marker is going to be explored. The findings demonstrate thatfunctional characteristics displayed by mouse IgG mAbs are notpredictive for a chimer (Table 2). The antibody GA101 (obinutuzumab)does not have significant complement activity see Herter et al (2013)Mol Cancer Ther. 12(10):2031-42. doi: 10.1158/1535-7163.MCT-12-1182.

Example 3

IgG1-CD20 monoclonal antibodies (mAbs) like rituximab, ofatumumab andobinutuzumab are used in the clinics to treat patients diagnosed withdifferent kinds of B-cell malignancies. For IgG1-CD20 mAbs variousmechanisms-of-action are known to contribute to the eradication of tumorcells:⁵⁶⁻⁶¹⁾ (a) antibody-dependent cytotoxicity and phagocytosis(ADCC/ADCP) mediated by the engagement of Fc gamma receptors (FcγR) onpredominantly natural killer cells and monocytes/macrophages; (b)activation of the classical complement pathway by binding C1q, resultingin tumor cell lysis by CDC; (c) induction of PCD upon direct binding toCD20 molecules. Additionally, it was suggested that the treatment withmAbs can induce an adaptive cellular immune response. Findings by Abèsand colleagues showed that an Fc-mediated response results in along-lasting protection of immunocompetent mice after tumorre-challenge.⁽⁶²⁾ The induction of cytotoxic T-cells might be initiatedby dendritic cells, which cross-present tumor-derived peptides afterphagocytosis of Ab-opsonized target cells.^((63,64)) This long-termanti-tumor immunity has not yet been shown in patients, but mightexplain why for some patients a durable tumor regression can be achievedwith CD20 mAbs.

The engagement of effector cells required for the induction of ananti-tumor response by IgG1-CD20 mAbs relies on the interaction withFcγR. However, single nucleotide polymorphisms (SNPs) within FcγIIa andFcγRIIIa have been associated with better or worse treatmentoutcomes.⁽⁶⁵⁻⁶⁸⁾ Furthermore, due to unknown reasons not all patientsrespond to IgG1-CD20 mAb therapy. Patients that relapse or developresistance after rituximab treatment have also been reported.⁽⁶⁹⁻⁷³⁾Thus, alternative treatment options are required to overcome theselimitations. One possibility is the use of a different Ab isotype,namely IgA. IgA is the second most prominent antibody in blood, afterIgG, and the predominant Ab at the mucosa. The monomeric version of IgAis mostly found in serum, whereas polymeric IgA is produced at mucosalsites. The 2 Ab subclasses, IgA1 and IgA2, differ structurally in theirhinge regions, which is 13 amino acids longer for IgA1 than for IgA2.This might enable an improved reach for antigens that are distant, butat the same time makes it more prone to degradation by proteases.⁽³⁶⁾Furthermore, the hinge region of IgA1 mAbs carries several O-linkedglycosylation sites, which are absent in IgA2 mAbs. IgA2 exists as 3allotypes; IgA2(m1) has 2 additional N-linked glycosylation sitescompared with IgA1, and IgA2(m2) and IgA2(n), which have 3 additionalN-linked glycosylation sites. Contrary to IgG, IgA is a weak activatorof the classical complement pathway as it cannot bind C1q.⁽³⁷⁾ However,IgA mAb have been shown to activate the complement system through thelectin pathway, as the carbohydrate recognition domain (CRD) ofmannan-binding lectin (MBL) can bind to IgA.⁽³⁸⁾

IgA engages immune effector cells by binding to the FcαRI (CD89), whichis expressed on cells of the myeloid lineage: neutrophils, monocytes,different macrophage populations and eosinophils.⁽³⁹⁾ Expression on invitro generated dendritic cells was shown,^((23, 24)) but remainscontroversial. Neutrophils express high levels of the FcαRI, whilemacrophages have lower expression.⁽⁴²⁾ In ADCC assays with IgA mAbstargeting solid tumor targets, neutrophils have been shown toefficiently eradicate tumor cells.⁽⁴³⁻⁴⁶⁾ In contrast, IgG1 mAbs wereless able to engage this effector cell population.Monocyte/macrophage-mediated tumor cell killing was shown to becomparable between IgA and IgG mAbs⁽⁴⁴⁾ Next to the activating FcγRIIIa,macrophages also express the inhibitory FcγRIIb. It has been shown thatthe presence of FcγRIIb reduces mAb activity.⁽⁴⁷⁾ For IgA, no inhibitoryreceptor has been described yet.

The knowledge on IgA mAb targeting tumor-associated antigens hasincreased significantly over the last few years. Several bottlenecksthat were faced a few years ago are now overcome, and are currently ableto produce and purify sufficient amounts of monomeric IgA mAbs for invitro and in vivo testing. Mice lack a receptor for IgA, therefore thegeneration of human FcαRI transgenic now allows in vivo testing.⁽⁴⁸⁾Boross and colleagues eventually showed in an immunocompetent tumormodel the great potential of IgA mAbs in a therapeutic setting.⁽⁴⁴⁾ Themajority of IgA mAbs studied so far are targeting Her2 or EGFR, antigensexpressed on solid tumors. Only one study has looked at the potential ofmonomeric IgA-CD20 mAbs.⁽⁴⁹⁾ Complement-mediated tumor cell killing wasdemonstrated to rely on weak indirect activation of the classicalpathway and more pronounced direct activation of the alternativepathway. With a passive immunization strategy in FcαRI transgenic mice,a good protection against tumor development with monomeric IgA2-CD20mAbs was achieved. However, therapeutic in vivo testing for IgA-CD20mAbs has not yet been performed. Further, a direct comparison of IgA1and IgA2 mAbs, in particular, with respect to their complementactivation properties is lacking. Therefore, the goal of the currentstudy was to generate several IgA-CD20 mAbs of the different subclassesand characterize them. Described here is the preliminary in vitrocharacterization of unique IgA1- and IgA2-CD20 mAbs. The previouslygenerated mIgG2c-CD20 mAbs m1 and m2 (Example 1) were selected to beproduced as IgA1 and IgA2 variants. Their preliminary in vitrocharacterization revealed promising potential.

Material and Methods

Antibodies

The following antibodies were used in experiments as positive ornegative controls: rituximab (RTX; Pharmacy UMC Utrecht),anti-hCD20-hIgA2 (IgA2-RTX; InvivoGen), anti-hCD20-hIgA1 (IgA1-RTX;InvivoGen); IgA1- and IgA2(m1)-Her2 (own production), ofatumumab (OFA,Pharmacy UMC Utrecht), B1 (mIgG2a-CD20 mAbs, kindly provided by MarkCragg, University Southhampton, UK), and trastuzumab (TRA, Pharmacy UMCUtrecht).

Cell Lines

The Burkitt lymphoma cell lines Ramos and Raji (ATCC) were maintained inRPMI culture medium containing RPMI-1640+HEPES+glutamine (Invitrogen)supplemented with 10% FCS and 1× P/S at 37° C./5% CO₂. EL4-CD20 cellswere generated as previously described⁽²³⁾ and cultured in RPMI culturemedium.

Production of IgA-CD20 mAbs

The variable heavy and light chain (HC and LC) sequences (synthesized byShineGene) were flanked by the HindIII and NotI restriction sites. Thevariable regions were cloned into the Lonza vectors pEE14.4-kappaLC,pEE14.4-IgA1, and pEE14.4-IgA2(m1).

For transient antibody production, the HEK FreeStyle™ 293F Cell(Invitrogen) system was used as previously described.⁽²²⁾ Briefly,HEK293F cells were transfected with LC, HC and pAdVAntage™ Vector(Promega) DNA diluted in Opti-MEM (Life Technologies) together with293fectin™ Transfection Reagent (Life Technologies). After 4 hours 1×P/S (Gibco) was added. Supernatant was harvested 4 days posttransfection, filtered and stored until purification at 4° C.

All column purification steps were performed using the ÄKTAPrime plussystem (GE Healthcare). The antibody containing supernatant was diluted1:1 with the binding buffer (1× PBS; Sigma-Aldrich) and IgA mAbs werepurified using a 5 mL HiTrap KappaSelect column (GE-Healthcare). Boundprotein was eluted from the column with 0.1 M Glycine, pH 2.5. Fractionswere collected, directly neutralized with 1 M Tris, pH 8.8 and pooledbased on protein concentration determined by A280 absorption measured ona Nanodrop. Protein containing fractions were subjected tosize-exclusion chromathography (SEC) using a HiPrep 26/60 SephacrylS-300 High Resolution column (GE Healthcare). Fractions containing themonomeric IgA mAbs were collected, pooled, and concentrated usingVivaSpin concentration columns (100 000 MWCO; Sartorius). Endconcentrations of the IgA mAbs were determined by Nanodrop using thefollowing formula:

$\frac{{OD}{value}{at}{280}{nm}}{{correction}{factor}\left( {{monomeric}{IgA}\text{?}} \right)}.$?indicates text missing or illegible when filed

IgG and IgA ELISA

A 96-well plate (NUNC maxisorp) was coated with goat-anti-huKappa(Southern Biotech) diluted in 1× PBS (Sigma-Aldrich) at 4° C. overnight.After blocking with 1% BSA/0.05% Tween-PBS and washing with 0.05%Tween-PBS, samples were added and incubated for 90 minutes at roomtemperature. Samples and standards were diluted in 1% BSA/0.05%Tween-PBS. Purified huIgA (Bethyl Lab) was used as a standard for IgAmAbs. Washed plates were incubated with goat-anti-human IgA-HRP(Southern Biotech) diluted in 0.05% Tween-PBS as secondary antibody for1 hour at room temperature. Round antibodies were detected with ABTS(Roche) and subsequent measurement at 405 nm with a Multiscan RC(Thermolab systems).

CD20 Binding Assay

10⁵ Ramos cells were plated out in a 96-wells plate and incubated withmAb diluted in 1× PBS for 45 minutes on ice. After extensive washing,bound Abs were detected by incubating the cells for 45 minutes on icewith goat-F(ab′)2-anti-humanIgA-RPE (Jackson ImmunoResearch). Cells werefixed with 1% paraformaldehyde before measuring them on the FACS CantoII (BD).

CDC Assay

10⁵ Daudi or Ramos cells were opsonized with mAbs diluted in RPMIculture medium for 30 minutes at room temperature. Next, 15.5% normalhuman serum (NHS) pooled from 8 donors was added and the mixtureincubated for different time spans (15, 60, 240, and 360 min) at 37° C.Serum for blocking experiments was treated before addition to the cellsby heat-inactivation (30 minutes at 56° C.), pre-incubation with 10 mMEGTA+2 mM MgCl₂, or addition of eculizumab (anti-C5 mAb) at a mAb:C5molar ratio of 5:1 for 15 minutes at room temperature. Cells were washedwith 1× PBS and dead cells stained with 7-AAD (BD Pharmingen) for 15minutes at room temperature. Samples were measured on a FACS CantoII(BD) and complement-dependent tumor cell lysis was determined as thepercentage of 7-AAD positive cells.

Cell Death Induction Assay

0.4×10⁵ EL4-CD20 cells were incubated with 1 μg/mL mAb for 24 hours at37° C. Cross-linking Abs (20 μg/mL rabbit F(ab′)₂-anti-hIgG; 50 μg/mLrabbit F(ab′)₂-anti-mIgG, and 20 μg/mL goat F(ab′)₂-anti-human serumIgA; Jackson) were added 30 minutes after incubation of the cells withthe CD20 mAbs. Samples were collected, washed with Annexin V bindingbuffer and incubated with a mix of AnnexinV-APC and 7-AAD (both BDPharmingen) for 15 minutes at room temperature. Cells were measured on aFACS Canto H and the extent of programmed cell death was determined as %AnnexinV⁺7-AAD⁺+% AnnexinV⁺ cells.

B-Cell Depletion Assay

All leukocytes were isolated from healthy donor blood collected inHeparine tubes by Histopaque-ficoll density gradient and subsequentlysis of residual erythrocytes using eryhtrocyte lysis buffer (155 Mmammonium chloride, 0.1 mM EDTA and 10 mM potassiumbicarbonate, pH 7.4).Isolated leukocytes were combined with antibody dilutions in a 96-wellplate at 0.2×10⁶ leukocytes/well and incubated for 3 hours at 37° C./5%CO₂. The supernatant was removed after centrifugation, cells washed withice-cold PBS/0.1% BSA (FACS buffer) and stained with an antibody mixcontaining mouse-anti-human CD19-APC (Biolegend), mouse-anti-humanCD11b-PE (BD Pharmingen), mouse-anti-human CD14-V500 (BD Pharmingen),mouse-anti-human CD56-AlexaFluor488 (clone B159, BD Pharmingen), andmouse-anti-human CD3-PerCp (Biolegend) for 30 minutes on ice. Afterwashing, cells were resuspended in FACS buffer containing Cyto/CalMultifluor Plus Violet Flow Cytometer Alignment Beads (ThermoScientific), and flow cytometry was performed using a BD FACS Canto™ II.

Human PMN ADCC

ADCC assays with ⁵¹Cr-labeled target cells were performed as previouslydescribed. Briefly, PMNs were isolated from healthy individuals(MiniDonorDienst UMC Utrecht) by Ficoll/Histopaque separation (GEHealthcare; Sigma-Aldrich). ⁵¹Cr-labeled Daudi cells were combined withPMNs(effector-to-target ratio=40:1) and CD20 mAb in dilution. After 4hours incubation at 37° C./5% CO₂, the supernatant was harvested andcounted in a liquid scintillation counter (MicroBeta; Perkin Elmer).Lysis was calculated using the following formula:

${\%{lysis}} = {\frac{{counts}_{sample} - {counts}_{{minimal}{release}}}{{counts}_{{maximum}{release}} - {counts}_{{minimal}{release}}} \times 100.}$

Daudi cells with effector cells in RPMI culture medium or in mediumsupplemented with 2.5% Triton X-100 (Roche Diagnostics) were used todetermine minimal and maximum release, respectively.

Results

Antibody Production and Purification

The IgA1 and IgA2 mAbs were produced by transfection of HEK293F cells.First, the optimal ratio between heavy chain, light chain and pAdvantageDNA was determined in a small scale test transfection. For all mAbs, a1:2:1 ratio to result in the highest production (FIG. 12A) was found.The new mAbs were subsequently produced by a linear upscaling approach.The full-size mAbs were isolated by a two-step purification protocol,including 1) anti-kappa purification (FIG. 12B) and 2) size-exclusionchromatography (SEC) (FIG. 12C). Whereas the kappa purification peakslook the same for both subclasses, a different pattern could be observedduring the SEC. Next to the full-size Abs, IgA1 samples contained higherlevels of loose kappa light chains (FIG. 12C left). In contrast, IgA2samples contained more Ab conjugates (shorter retention time; FIG. 12Cright). The yields of full-size mAbs obtained after SEC were comparablebetween both subclasses. Purity and integrity of the purified IgA mAbswas assessed by SDS-PAGE (data not shown).

Functional Characterization

First, the ability of the antibodies to bind to CD20-expressing Ramoscells (FIG. 13 ) was assessed. Binding capacity was in the same rangefor all IgA2-CD20 mAbs. However, IgA1 mAbs had a better binding capacitycompared to the IgA2 mAbs.

Next, the functionality of the newly synthesized antibodies was tested.A subset of IgG-CD20 mAbs, known as Type II CD20 mAbs, induces PCD uponbinding to their target. The IgA mAbs was subjected to a cell deathinduction assay. The incubation of EL4-CD20 tumor cells with the mAbsfor 24 hours led to a marginal increase of PCD by the new IgA-CD20 mAbs(FIG. 14 ). This was slightly more pronounced for IgA1 than IgA2 mAbs.

Subsequently, the ability of IgG1- and IgA-CD20 mAbs was compared toinduce CDC. Complement-mediated lysis of Daudi cells was observedalready after 15 minutes with IgG1-CD20 mAbs, but not with IgA-CD20 mAbs(FIG. 15A). Minor CDC induction by IgA-CD20-1 mAbs was seen after 60minutes incubation with complement active serum, which increased withlonger incubation times. Unexpectedly, Ramos cells, which express lessCD20, but higher levels of CD46 and CD59 than Daudi cells, were moresusceptible to CDC (FIG. 15B). Furthermore, IgA1-CD20complement-mediated lysis was delayed but reached a comparable level asIgA2-CD20 mAbs after 240 minutes. To confirm that the IgA-CD20-1 inducedlysis was mediated by complement activation, CDC assays were performedin the presence of different complement inhibitors (FIG. 15C). The lysisinduced by IgA-CD20-1 mAbs was abolished when heat-inactivated serum andserum pre-treated with eculizumab (anti-C5 mAb), was used. To determineif the alternative pathway is engaged by IgA mAbs, EGTA+MgC₂ was used toinhibit the classical and lectin pathway. Lysis was reduced tobackground levels not only for IgG1, but also by IgA-CD20-1 mAbs.

After determining the ability of the new IgA-CD20 mAbs to inducePMN-mediated tumor cell lysis in a chromium release assay, the potentialof the IgA-CD20 mAbs to deplete human B cells was then analyzed.Incubation of leukocytes from healthy donors with the mAbs resulted in aconcentration-dependent decline of CD19⁺ events (FIG. 16A). This effectwas comparable between RTX and the IgA-CD20 mAbs. The number of CD19⁺events (FIG. 16B) steadily decreased for all IgA-CD20 mAbs and reached aplateau between 0.05 and 0.5 μg/mL mAbs. In contrast, the number ofCD19⁺ events upon RTX incubation reached a minimum at 0.05 μg/mL, butincreased at higher concentrations again. For both isotypes, the numberof CD19′ events reached an optimum between 0.05 to 0.5 μg/mL (FIG. 16C).

Discussion

At the moment, all CD20 targeting mAbs on the market are of the IgG1isotype. The varying response to the treatment stresses the need todevelop alternative therapies. In the recent years, researchers haveinvestigated the anti-cancer potential of IgA mAbs targeting, inparticular, solid tumor targets like Her2 and EGFR.^((43-46, 74)) Here,the comparison of IgA1-CD20 and IgA2(m1)-CD20 mAbs is described, whichhave unique variable domain sequences of two previously selected mousemAbs. Functional characterization of the parental mouse mAbs m1 and m2showed distinct behavior in their complement-mediated tumor cell lysiscapacity, binding kinetics and epitope recognition (Example 1).

Fab- and Fc-mediated mechanisms were studied leading to the eradicationof tumor cells as described for IgG1 mAbs. Some IgG1-CD20 mAbs (e.g.,Obinutuzumab and B1) elicit tumor cell killing upon binding to CD20(Fab-mediated effect). PCD with IgG1-CD20-1 was observed, but notIgG1-CD20-2. In contrast, both IgA-CD20-1 and IgA-CD20-2 induced PCD,albeit at a low level, indicating that this mechanism-of-action for IgAmAbs is independent of the epitope. To strengthen this hypothesis, otherknown antibodies that differ in their PCD capacity as IgG1 and targetdifferent epitopes (e.g., RTX and Obinutuzumab) would need to becompared as IgA variants.

CDC induced by IgG1 is an efficient Fc-mediated mechanism to lyse tumorcells. IgG1-CD20 mAbs activate the classical complement pathway bybinding C1q. However, the activation of this pathway is unlikely for IgAmAbs as they lack the C1q recognition site.⁽³⁷⁾ In literature it wasdescribed that IgA Abs isolated from human serum bind MBL.⁽³⁸⁾ Thisresulted in C3 deposition, indicating the engagement of the lectinpathway. Still, Pascal and colleagues showed that IgA2-CD20 mAbs lyse asubset of CD20⁺ tumor cell lines by directly activating the alternativeand indirectly engaging the classical pathway.⁽³⁹⁾ With the data, theinvolvement of CDC in tumor cell killing by IgA mAbs was confirmed.However, the results exclude activation of the alternative pathway. Tofurther dissect the contribution of the different complement pathways,additional experiments have to be performed with specific inhibitors.For example, C1q depleted serum can be used to block the classicalpathway, Factor B depleted serum for inhibition of the alternativepathway, and MASP-1 or MASP-2-specific inhibitors to block the lectinpathway.^((75, 76)) For the first time, both subclasses next to eachother were compared and it showed that IgA2 mAbs are faster at inducingCDC than IgA1 mAbs. This discrepancy might be a consequence of thedifferential glycosylation pattern of the heavy chain of IgA1 (2N-linked glycans) and IgA2(m1)(4 N-linked glycans), resulting in abetter activation of the lectin pathway by IgA2.

Properties like target antigen expression level, expression of membranebound complement-regulatory proteins, the epitope, and binding kineticshave been implicated to influence IgG1-mediated CDC. Thus far, only arole for the epitope of IgA mAbs, as IgA-CD20-1 mAbs, could beestablished, derived from mIgG with better CDC activity, also activatecomplement better than IgA-CD20-2 mAbs. Whether the other properties arealso important, needs to be elucidated.

ADCC by FcαRI expressing effector cells has been shown to be mediatedfaster by PMNs than by monocytes/macrophages. To determine the role ofthese immune effector cells in IgA-CD20-mediated B-cell depletion, CD19⁺B cells were depleted in an autologous setting in the absence of acomplement source. mAb-mediated depletion of CD19⁺ cells was generallybetter for IgA-CD20 mAbs than for RTX, with IgA1-CD20 mAbs performingthe best. However, both isotypes induce the loss of CD19 expression.This is in line with previously published findings that B cells fromhealthy donors incubated with RTX lose their CD19 expression withoutundergoing cell death.⁽⁵²⁾ The same antigenic modulation was describedfor CD20, with 75-90% reduction in less than 45 minutes.⁽⁵⁵⁾ Neithershedding nor internalization were implicated in the loss of CD19.Instead, shaving/trogocytosis in an Fc-dependent manner, particularlymediated by neutrophils and monocytes, was described as themechanism-of-action. It is speculated that the stronger engagement ofPMNs by IgA mAbs compared to IgG1 mAbs eventually leads to tumor celldeath by trogocytosis.⁽⁵³⁻⁵⁴⁾

The therapeutic dose of RTX should be carefully determined, as too highconcentrations were shown to decrease Ab efficacy in the in vitro B-celldepletion assay. In contrast, this effect was not observed with IgA-CD20mAbs. Nonetheless, as CD20 mAbs modulate the expression of B-cellmarkers, B-cell tracking might represent a better approach to properlydetermine mAb-mediated B-cell depletion.

Example 4 Characteristics of IgA CD20 Antibodies.

The human body uses IgA do defend itself against pathogens,predominantly on the mucosa barrier. It kills pathogens either viadirect mechanisms or via neutrophil activation. Very low quantities ofIgA can effectively trigger neutrophils via the Fcα receptor (FcαR).Importantly, neutrophils are far more effective killer cells than NKcells and possess excellent tissue penetration capacity. Thus, IgA seemsan attractive antibody class for tumor immunotherapy. Nevertheless, itis difficult to develop clinical IgA antibodies. Until recently, therewere no good in vivo model systems for IgA immunotherapy since mice lackexpression of the FcαR. The research group has developed a transgenicmice that expresses human FcαR (CD89) on neutrophils. With this mousemodel, it is demonstrated that therapeutic IgA antibodies caneffectively defend mice against tumor outgrowth in several in vivomodels, long-term and short-term on different locations, and inimmunocompetent mice6. Using a unique locally developed immunizationmethod, a broad panel of novel CD20 antibodies (all IgG) was obtained.Two of these antibodies have clinical potential, killing tumor cells invitro more effectively than all currently available clinical CD20antibodies. Both antibodies have now been transformed into chimericantibodies with human IgA Fc fragment (+/−70% huIgA) and currentlyexplored for their efficacy (FIG. 17 ).

CD20 Internalization

It has been described in references 77-79 that certain antibodies thatare used therapeutically in lymphoma treatment (eg, rituximab andofatumumab) undergo Fcγ receptor IIb (FcγRIIb)-mediated internalizationfrom the B-cell surface with important implications for antibody-basedtherapeutics.

FcγR expression on target cells themselves is critical for this Ab-CD20complex internalization. Immune effector cell Fc-FcγR interactions intrans act to deplete the target cell, particularly when the antigen isexpressed at high levels. Although the importance of activating FcγR forthe efficacy of rituximab in xenograft models was demonstrated before,now it is known that the inhibitory FcR, FcγRIIb, expressed on certaintypes of B-cell malignancies, also plays a role in Ab-CD20 complexinternalization. FcγRIIb contains an immunoreceptor tyrosine-basedinhibitory motif that recruits specific phosphatases and acts to opposesignaling by activating FcγRs. FcγRIIb-mediated internalization ofrituximab from the B-cell surface was shown to limit effector cellengagement, an effect that may be particularly important in malignanciessuch as chronic lymphocytic leukemia and mantle cell lymphoma thatexpress high levels of FcγRIIb and show reduced responses to CD20 mAbtherapy.

This effect was not seen with the IgA antibodies of antibodies m1, m2,m7, m9, m10. These do not have this negative side effect. Indeed, when Bcells were isolated from blood of healthy volunteers, CD20 IgGantibodies show a decrease of the CD20 target molecule of 40 to 60percent, whereas with the IgA antibodies remained at a level of 80/6 onthe cell surface of B cells (see FIGS. 25, 26.1, 26.2 and 26.3 ),suggesting that IgA antibodies have a diminished side effect on therapycompared to IgG antibodies.

Methods for CD20 Internalization

PBMC were separated from healthy donors by ficoll (GE healthcare)density gradient and used for B cell isolation using MACS human B cellisolation kit H (Milteny biotec). 5×10e4 isolated B cells were seeded inconical 96 wells plates and incubated with 1 ug/ml AlexaFluor 488(AL488) labeled (Molecular Probes) Ab for 3 hours at 37° C./5% CO2.Unbound Ab was washed away and the AL488 signal quenched using 25 ug/mlgoat-anti-AL488 (life technologies). % remaining CD20 is calculated as((MFI unquenched−MFI quenched)/MFI unquenched)×100.

Example 5 Materials and Methods

ADCC Assay

ADCC assays with ₅₁Cr-labeled target cells were performed as previouslydescribed.⁽² 2) Briefly, PBMC and PMN isolated from healthy individuals(MiniDonorDienst UMC Utrecht) by Ficoll/Histopaque separation (GEHealthcare) were combined with 51Cr-labeled Daudi cells(effector-to-target ratio=100:1) and CD20 mAb in dilution. After 4 hoursincubation at 37° C./5% CO2, the supernatant was harvested and countedin a liquid scintillation counter (MicroBeta; Perkin Elmer). Lysis wascalculated using the following formula: % lysis=((counts ofsample−minimum release)/(maximum release−minimum release))×100. Daudicells with effector cells in culture medium or in medium supplementedwith 5% Triton X-100 (Roche Diagnostics) were used to determine minimumand maximum release, respectively.

EL4-CD20 Lymphoma Model

C57BL/6 mice were purchased from Janvier (France) or bred in thefacilities. 4-6 mice/group were injected intraperitoneally (i.p.) with5×105 CellTraceViolet (10 μM, Invitrogen)-labeled EL4-CD20 cells. After16 hours, mice were treated with mAb or PBS (100 μl injected i.p. Aperitoneal lavage with PBS containing 5 mM EDTA was performed after 24hours and the amount of remaining tumor cells was determined usingTruCount tubes (BD). All experiments were approved by the Animal EthicalCommittee of the UMC Utrecht.

Saturation of CD20 on EL4-CD20 Lymphoma Cells

After performing the previously described EL-4 lymphoma model, collectedtumor cells were washed twice with PBS and stained with secondary IgA orIgG-PE labelled antibody. Saturation of CD20 with anti-CD20 antibody wasshown if no increased signal was present after adding antibody (10μg/ml) ex vivo to the cells before staining with secondary antibody.Shaving of CD20 was determined by comparing CD20 expression on EL4 cellsfrom PBS treated mice against antibody treated mice.

Results

Functional Characterization:

The ability of the antibodies to bind to CD20-expressing Ramos cells andthe functionality of the antibodies on PCD are depicted in FIG. 13 andFIG. 14 . The ability of IgG1- and IgA-CD20 mAbs to induce CDC isdepicted in FIGS. 15A-15C (see also Example 3). The ability of the newIgA-CD20 mAbs to induce PMN-mediated tumor cell lysis in a chromiumrelease assay is depicted in FIGS. 27A, 27B. IgA1 antibodies do havesimilar ADCC potential, but IgA1-CD20 UMAB007 is the most effective(FIG. 27A). For IgA2 antibodies, all antibodies show a similareffectivity, but IgA2-CD20 UMAB010 has the highest killing potential(FIG. 27B). Next, the ability of the IgA-CD20 mAbs to deplete human Bcells was analyzed. Incubation of leukocytes from healthy donors withthe mAbs resulted in a concentration-dependent decline of CD19+ events(FIG. 16A). This effect was comparable between RTX and the IgA-CD20mAbs. The number of CD19− events (FIG. 16B) steadily decreased for allIgA-CD20 mAbs and reached a plateau between 0.05 and 0.5 μg/mL mAbs. Incontrast, the number of CD19+ events upon RTX incubation reached aminimum at 0.05 μg/mL, but increased 5 at higher concentrations again.For both isotypes, the number of CD19- events reached an optimum between0.05 to 0.5 μg/mL (FIG. 16C). The in vivo efficacy of a selection ofthese antibodies was studied in the EL4-CD20 lymphoma model (FIGS.28A-28C). IgA2-CD20 UMAB002 and IgA1-CD20 UMAB007 were as potent asrituximab, which shows the potential of IgA antibodies in vivo. Finally,it was shown that loss of CD20 expression was seen after treatment withrituximab (FIG. 28A), but that expression is not lowered after treatmentwith IgA antibodies (FIGS. 28B, 28C). Next the CD20 expression of theEL4-CD20 cells was determined in the samples. It is shown that CD20expression is lost after IgG treatment, but not after IgA treatment invivo (FIG. 29 ). Umab 002 and 007 seem to perform better than umab 001in this particular assay. IgA2 appears to be the better isotype for umab001 and umab 002, while IgA1 is better in combination with umab 007.IgA1-CD20 UMAB . . . where the dots indicate three digits is an antibodywith the indicated constant region and a variable domain of one of thefive specified murine antibody. The last digit of the UMAB referenceindicates the variable domain of the m antibody with the same digit.

TABLE 1 Panel of novel CD20 mIgG antibodies. sequencing mouse # nameisotype HC LC purified 1 m1 IgG2c x x x m2 IgG2c x x x m3 IgG2b x x x m4IgG2b x x x m5 IgG2b x x x m6 IgG2b x x x m7 IgG2b x x x 2 m8 IgG2b x xm9 IgG2c x x x 3 m10 IgG2c x x x m11 IgG2b x x 4 m12 unknown x 5 m13IgG2c x x m14 IgG2c x m15 IgG2c x x 6 m16 IgG3 x x m17 IgG2b x x x

TABLE 2 Functionality in vitro Name CDC ADCC PCD m1 (mIgG2c) +++ ++ −IgG1-CD20-1 + + ++ m2 (mIgG2c) + ++ − IgG1-CD20-2 +/− +++ − m7 (mIgG2b)+++ − − IgG1-CD20-7 +++ ++ − m9 (mIgG2c) +++ ++ − IgG1-CD20-9 ++ +++ −m10 (mIgG2c) ++ ++ − IgG1-CD20-10 + +++ −

TABLE 3 Affinity values of CD20 mAbs. Values were obtained by fitting aOneToOne binding model to the binding curves that had an associationtime of one hour and a dissociation time of three hours. The affinity(K_(D)) is obtained from the ratio between the dissociation rateconstant (k_(off)) and the association rate constant (k_(on)).non-competitive competitive k_(on) (1/M*s) k_(off) (1/s) K_(D) (nM)k_(on) (1/M*s) k_(off) (1/s) K_(D) (nM) m1 3.73E+04 3.91E-05 1.052.61E+04 6.92E-05 2.65 m2 8.54E+04 1.77E-05 0.21 8.17E+04 7.80E-05 0.96m9 4.57E+04 1.13E-05 0.25 5.39E+04 6.53E-06 0.12 m10 4.42E+04 8.17E-060.19 5.57E+04 2.82E-05 0.51 OFA 5.01E+04 5.71E-06 0.11 7.70E+04 3.06E-050.40 RTX 5.14E+04 3.05E-05 0.59 7.35E+04 5.50E-05 0.75

CITED ART

-   Hallek M. et al. Addition of rituximab to fludarabine and    cyclophosphamide in patients with chronic lymphocytic leukaemia: a    randomised, open-label, phase 3 trial. Lancet 376:1164-1174 (2010).-   2. Coiffier B. et al. Long-term outcome of patients in the LNH-98.5    trial, the first randomized study comparing rituximab-CHOP to    standard CHOP chemotherapy in DLBCL patients: a study by the Groupe    d'Etudes des Lymphomes de l'Adulte. Blood 116:2040-2045 (2010).-   3. Keating G. M. Rituximab: a review of its use in chronic    lymphocytic leukaemia, low-grade or follicular lymphoma and diffuse    large B-cell lymphoma. Drugs 70:1445-1476 (2010).-   4. Rastetter, W., A. Molina and C. A. White. Rituximab: expanding    role in therapy for lymphomas and autoimmune diseases. Annu. Rev.    Med. 55:477-503 (2004).-   5. Badin, F. and J. Hayslip. Rituximab in the treatment of B-cell    non-Hodgkin lymphoma, focus on outcomes and comparative    effectiveness. Clinicoecon Outcomes Res. 2:37-45 (2010).-   6. Sandhu, S. and S. P. Mulligan. Ofatumumab and its role as    immunotherapy in chronic lymphocytic leukemia. Haematologica    100:411-414 (2015).-   7. Laurenti L., I. Innocenti, F. Autore, S. Sica and D. G. Efremov.    New developments in the management of chronic lymphocytic leukemia:    role of ofatumumab. Onco Targets Ther. 9:421-429 (2016).-   8. Goede V. et al. Obinutuzumab plus chlorambucil in patients with    CLL and coexisting conditions. N. Engl. J. Med. 370:1101-1110    (2014).-   9. Sehn L. H. et al. GADOLIN: Primary results from a phase III study    of obinutuzumab plus bendamustine compared with bendamustine alone    in patients with rituximab-refractory indolent non-Hodgkin lymphoma.    Journal of Clinical Oncology, 2015 ASCO Annual Meeting Vol 33, No    15_suppl (May 20 Supplement) (2015).-   10. Cragg M. S. et al. Complement-mediated lysis by anti-CD20 mAb    correlates with segregation into lipid rafts. Blood 101:1045-1052    (2003).-   11. Chan H. T. et al. CD20-induced lymphoma cell death is    independent of both caspases and its redistribution into triton    X-100 insoluble membrane rafts. Cancer Res. 63:5480-5489 (2003).-   12. Mossner E. et al. Increasing the efficacy of CD20 antibody    therapy through the engineering of a new type II anti-CD20 antibody    with enhanced direct and immune effector cell-mediated B-cell    cytotoxicity. Blood 115:4393-4402 (2010).-   13. Beers S. A. et al. Type II (tositumomab) anti-CD20 monoclonal    antibody out performs type I (rituximab-like) reagents in B-cell    depletion regardless of complement activation. Blood 112:4170-4177    (2008).-   14. Teeling J. L. et al. Characterization of new human CD20    monoclonal antibodies with potent cytolytic activity against    non-Hodgkin lymphomas. Blood 104:1793-1800 (2004).-   15. Stanglmaier M., S. Reis and M. Hallek. Rituximab and alemtuzumab    induce a nonclassic, caspase-independent apoptotic pathway in    B-lymphoid cell lines and in chronic lymphocytic leukemia cells.    Ann. Hematol. 83:634-645 (2004).-   16. Bornstein G. G. et al. Development of a new fully human    anti-CD20 monoclonal antibody for the treatment of B-cell    malignancies. Invest. New Drugs 28:561-574 (2010).-   17. Du J. et al. Structural basis for recognition of CD20 by    therapeutic antibody Rituximab. J. Biol. Chem. 282:15073-15080    (2007).-   18. Niederfellner G. et al. Epitope characterization and crystal    structure of GA101 provide insights into the molecular basis for    type I/II distinction of CD20 antibodies. Blood 118:358-367 (2011).-   19. Polyak M. J. and J. P. Deans. Alanine-170 and proline-172 are    critical determinants for extracellular CD20 epitopes; heterogeneity    in the fine specificity of CD20 monoclonal antibodies is defined by    additional requirements imposed by both amino acid sequence and    quaternary structure. Blood 99:3256-3262 (2002).-   20. Teeling J. L. et al. The biological activity of human CD20    monoclonal antibodies is linked to unique epitopes on CD20. J.    Immunol. 177:362-371 (2006).-   21. Goldenberg D. M., F. Morschhauser and W. A Wegene. Veltuzumab    (humanized anti-CD20 monoclonal antibody): characterization, current    clinical results, and future prospects. Leuk. Lymphoma 51:747-755    (2010).-   22. Meyer S. et al. Improved in vivo anti-tumor effects of IgA-Her2    antibodies through half-life extension and serum exposure    enhancement by FcRn targeting. MAbs 8:87-98 (2016).-   23. DiGaetano N. et al. Complement activation determines the    therapeutic activity of rituximab in vivo. J. Immunol 171-1581-1587    (2003).-   24. Congdon E. E., J. Gu, H B R Sait and F M. Sigurdsson. Antibody    Uptake into Neurons Occurs Primarily via Clathrin-dependent Fcγ    Receptor Endocytosis and Is a Prerequisite for Acute Tau Protein    Clearance*. J. Biol. Chem. 288:35452-35465 (2013).-   25. Slootstra J. W., W. C. Puijk, G. J. Ligtvoet, J. P. Langeveld    and R. H. Meloen,. Structural aspects of antibody-antigen    interaction revealed through small random peptide libraries. Mol.    Divers. 1:87-96 (1996).-   26. Timmerman P., J. Beld, W. C. Puijk, and R. H. Meloen. Rapid and    quantitative cyclization of multiple peptide loops onto synthetic    scaffolds for structural mimicry of protein surfaces. Chembiochem.    6:821-824 (2005).-   27. Boross P. et al. The in vivo mechanism of action of CD20    monoclonal antibodies depends on local tumor burden. Haematologica    96:1822-1830 (2011).-   28. Esser C. and A. Radbruch. Immunoglobulin class switching:    molecular and cellular analysis. Annu. Rev. Immunol. 8:717-735    (1990).-   29. Klein C. et al. Epitope interactions of monoclonal antibodies    targeting CD20 and their relationship to functional properties. MAbs    5:22-33 (2013).-   30. Umana P. et al. Novel 3rd Generation Humanized Type II CD20    Antibody with Glycoengineered Fc and Modified Elbow Hinge for    Enhanced ADCC and Superior Apoptosis Induction. 11:108 (2006).-   31. Withoff S. et al. Characterization of BIS20×3, a bi-specific    antibody activating and retargeting T-cells to CD20-positive    B-cells. Br. J. Cancer 84:1115-1121 (2001).-   32. Goldenberg D. M. et al. Properties and structure-function    relationships of veltuzumab (hA20), a humanized anti-CD20 monoclonal    antibody. Blood 113:1062-1070 (2009).-   33. Li B. et al. Characterization of a rituximab variant with potent    antitumor activity against rituximab-resistant B-cell lymphoma.    Blood 114:5007-5015 (2009).-   34. Diebolder C. A. et al. Complement is activated by IgG hexamers    assembled at the cell surface. Science 343:1260-1263 (2014).-   35. de Jong R. N. et al. A Novel Platform for the Potentiation of    Therapeutic Antibodies Based on Antigen-Dependent Formation of IgG    Hexamers at the Cell Surface. PLoS Biol. 14, e1002344 (2016).-   36. Senior B W, J M Woof. The influences of hinge length and    composition on the susceptibility of human IgA to cleavage by    diverse bacterial IgA1 proteases. J Immunol 2005; 174: 7792-9.-   37 Rakema J E, M van Egmond. Immunoglobulin A: A next generation of    therapeutic antibodies? MAbs 2011; 3: 352-61.-   38. Roos A, L H Bouwman, D J van Gijlswijk-Janssen, M C Faber-Krol,    G L Stahl, M R Daba. Human IgA activates the complement system via    the mannan-binding lectin pathway. J Immunol 2001; 167: 2861-8.-   39. Otten M A, M van Egmond. The Fc receptor for IgA (FcalphaRI,    CD89). Immunol Lett 2004; 92: 23-31.-   40. Geissmann F, P Launay, B Pasquier, Y Lepelletier, M Leborgne,    Lehuen A, et al. A subset of human dendritic cells expresses IgA Fc    receptor (CD89), which mediates internalization and activation upon    cross-linking by IgA complexes. J Immunol 2001; 166: 346-52.-   41. Heystek H C, C Moulon, A M Woltman, P Garonne, C van Kooten.    Human immature dendritic cells efficiently bind and take up    secretory IgA without the induction of maturation. J Immunol 2002;    168: 102-7.-   42. Hamre R, I N Farstad, P Brandtzaeg, H C Morton. Expression and    modulation of the human immunoglobulin A Fc receptor (CD89) and the    FcR gamma chain on myeloid cells in blood and tissue. Scand J    Immunol 2003; 57: 506-16.-   43. Lohse S, C Brunke, S Derer, M Peipp, P Boross, C Kellner, el al.    Characterization of a mutated IgA2 antibody of the m(1) allotype    against the epidermal growth factor receptor for the recruitment of    monocytes and macrophages. J Biol Chem 2012; 287: 25139-50.-   44. Boross P, S Lohse, M Nederend, J H Jansen, G van Tetering, M    Dechant, et al. IgA EGFR antibodies mediate tumour killing in vivo.    EMBO Mol Med 2013; 5: 1213-26.-   45. Meyer S, M Nederend, J H Jansen, K R Reiding, S R Jacobino, J    Meeldijk, el al. Improved in vivo anti-tumor effects of IgA-Her2    antibodies through half-life extension and serum exposure    enhancement by FcRn targeting. MAbs 2016; 8: 87-98.-   46. Rouwendal G J A, M M van der Lee, S Meyer, K R Reiding, J    Schnuten, G de Roo, et al. A comparison of anti-HER2 IgA and IgG1 in    vivo efficacy is facilitated by high N-glycan sialylation of the    IgA. mAbs 2016.-   47. R A Clynes, T L Towers, L G Presta, J V Ravetch. Inhibitory Fc    receptors modulate in vivo cytotoxicity against tumor targets. Nat    Med 2000; 6: 443-6.-   48. van Egmond M, A J van Vuuren, H C Morton, A B van Spriel, L    Shen, F M Hofhuis, et al. Human immunoglobulin A receptor    (FcalphaRI, CD89) function in transgenic mice requires both FcR    gamma chain and CR3 (CD11b/CD18). Blood 1999; 93: 4387-94.-   49. Pascal V, B Laffleur, A Debin, A Cuvillier, M van Egmond, D    Drocourt, et al. Anti-CD20 IgA can protect mice against lymphoma    development: evaluation of the direct impact of IgA and cytotoxic    effector recruitment on CD20 target cells. Haematologica 2012; 97:    1686-94.-   50. Bologna L, E Gotti, M Manganini, A Rambaldi, T Intermesoli, M    Introna, et al. Mechanism of action of type II, glycoengineered,    anti-CD20 monoclonal antibody GA101 in B-chronic lymphocytic    leukemia whole blood assays in comparison with rituximab and    alemtuzumab. J Immunol 2011; 186: 3762-9.-   51. Bologna L, E Gotti, R F Da, T Intermesoli, A Rambaldi, M    Introna, et al. Ofatumumab is more efficient than rituximab in    lysing B chronic lymphocytic leukemia cells in whole blood and in    combination with chemotherapy. J Immunol 2013; 190: 231-9.-   52. Jones J D, B J Hamilton, W F Rigby. Rituximab mediates loss of    CD19 on B cells in the absence of cell death. Arthritis Rheum 2012;    64: 3111-8.-   53. Homer H, C Frank, C Dechant, R Repp, M Glennie, M Herrmann, et    al. Intimate cell conjugate formation and exchange of membrane    lipids precede apoptosis induction in target cells during    antibody-dependent, granulocyte-mediated cytotoxicity. J Immunol    2007; 179: 337-45.-   54. Zhao X W. Targeting CD47-SIRPα interactions for potentiating    therapeutic antibody-mediated tumor cell destruction by phagocytes.    2014.-   55. Beum P V, E M Peek, M A Lindorfer, F J Beurskens, P J    Engelberts, P W Parren, et al. Loss of CD20 and bound CD20 antibody    from opsonized B cells occurs more rapidly because of    trogocytosis-mediated by Fc receptor-expressing effector cells than    direct internalization by the B cells. J Immunol 2011; 187-3438-47-   56. Glennie M J, R R French, M S Cragg, R P Taylor. Mechanisms of    killing by anti-CD20 monoclonal antibodies. Mol Immunol 2007; 44:    3823-37.-   57. Weiner G J. Rituximab: mechanism of action. Semin Hematol 2010;    47: 115-23.-   58. Lim S H, S A Beers, R R French, P W Johnson, M J Glennie M J, M    S Cragg. Anti-CD20 monoclonal antibodies: historical and future    perspectives. Haematologica 2010; 95: 135-43.-   59. Golay J, M Introna. Mechanism of action of therapeutic    monoclonal antibodies: promises and pitfalls of in vitro and in vivo    assays. Arch Biochem Biophys 2012; 526: 146-53.-   60. Boross P, J H Leusen. Mechanisms of action of CD20 antibodies.    Am J Cancer Res 2012; 2: 676-90.-   61. Okroj M, A Osterborg, A M Blom. Effector mechanisms of anti-CD20    monoclonal antibodies in B cell malignancies. Cancer Treat Rev 2013;    39: 632-9-   62. Abes R, E Gelize, W H Fridman, J L Teillaud. Long-lasting    antitumor protection by anti-CD20 antibody through cellular immune    response. Blood 2010; 116: 926-34.-   63. Weiner L M, M V Dhodapkar, S Ferrone. Monoclonal antibodies for    cancer immunotherapy. Lancet 2009; 373: 1033-40.-   64. Rafiq K, A Bergtold, R Clynes. Immune complex-mediated antigen    presentation induces tumor immunity. J Clin Invest 2002; 110: 71-9.-   65. Paiva M, H Marques, A Martins, P Ferreira, R Catarino, R    Medeiros. FcgammaRIIa polymorphism and clinical response to    rituximab in non-Hodgkin lymphoma patients. Cancer Genet Cytogenet    2008; 183: 35-40.-   66. Zhang W, M Gordon, A M Schultheis, D Y Yang, F Nagashima, M    Azuma, et al. FCGR2A and FCGR3A polymorphisms associated with    clinical outcome of epidermal growth factor receptor expressing    metastatic colorectal cancer patients treated with single-agent    cetuximab. J Clin Oncol 2007; 25: 3712-8.-   67. Musolino A, N Naldi, B Bortesi, D Pezzuolo, M Capelletti, G    Missale et al. Immunoglobulin G fragment C receptor polymorphisms    and clinical efficacy of trastuzumab-based therapy in patients with    HER-2/neu-positive metastatic breast cancer. J Clin Oncol 2008; 26:    1789-96.-   68. Dall'Ozzo S, S Tartas, G Paintaud, G Carron, P Colombat, P    Bardos, et at. Rituximab-dependent cytotoxicity by natural killer    cells: influence of FCGR3A polymorphism on the concentration-effect    relationship. Cancer Res 2004; 64: 4664-9.-   69. Rezvani A R, D G Maloney. Rituximab resistance. Best Pract Res    Clin Haematol 2011; 24: 203-16.-   70. Small G W, H L McLeod, K L Richards. Analysis of innate and    acquired resistance to anti-CD20 antibodies in malignant and    nonmalignant B cells. Peer J 2013; 1: e31.-   71. McLaughlin P, A J Grillo-Lopez, B K Link, R Levy, M S Czuczman,    M E Williams, et al. Rituximab chimeric anti-CD20 monoclonal    antibody therapy for relapsed indolent lymphoma: half of patients    respond to a four-dose treatment program. J Clin Oncol 1998; 16:    2825-33.-   72. Davis T A, A J Grillo-Lopez, C A White, P McLaughlin, M S    Czuczman, B K Link, el al. Rituximab anti-CD20 monoclonal antibody    therapy in non-Hodgkin's lymphoma: safety and efficacy of    re-treatment. J Clin Oncol 2000; 18: 3135-43.-   73. Kiss F, J Buslig, I Szegedi, B Scholtz, J Kappelmayer, C Kiss.    Early relapse after rituximab chemoimmunotherapy. Pediatr Blood    Cancer 2008; 50: 372-5.-   74. Dechant M, T Beyer, T Schneider-Merck, W Weisner, M Peipp, J G    van de Winkel, et al Effector mechanisms of recombinant IgA    antibodies against epidermal growth factor receptor. J Immunol 2007;    179: 2936-43.-   75. Kocsis A, K A Kekesi, R Szasz, B M Vegh, J Balczer, J Dobo, et    al. Selective inhibition of the lectin pathway of complement with    phage display selected peptides against mannose-binding    lectin-associated serine protease (MASP)-1 and -2: significant    contribution of MASP-1 to lectin pathway activation. J Immunol 2010;    185: 4169-78.-   76. Dunkelberger J R, W C Song. Complement and its role in innate    and adaptive immune responses. Cell Res 2010; 20: 34-50.-   77. Tipton T R, A Roghanian, R J Oldham, M J Carter, K L Cox, C I    Mockridge, R R French, L N Dahal, P J Duriez, P G Hargreaves, M S    Cragg, S A Beers. Blood. 2015 Mar. 19; 125(12):1901-9. doi:    10.1182/blood-2014-07-588376. Epub 2015 Jan. 28, Inhibitory FcγRIIb    (CD32b) becomes activated by therapeutic mAb in both cis and trans    and drives internalization according to antibody specificity.-   78. Vaughan A T, C Iriyama, S A Beers, C H Chan, S H Lim, E L    Williams, V Shah, A Roghanian, B Frendeus, M J Glennie, M S Cragg.    Blood. 2014 Jan. 30; 123(5):669-77. doi:    10.1182/blood-2013-04-490821. Epub 2013 Nov. 13. Fc gamma receptor    IIb on target B cells promotes rituximab internalization and reduces    clinical efficacy.-   79. Lim S H, A T Vaughan, M Ashton-Key, E L Williams, S V Dixon, H T    Chan, S A Beers, R R French, K L Cox, A J Davies, K N Potter, C I    Mockridge, D G Oscier, P W Johnson, M S Cragg, M J Glennie. Blood.    2011 Sep. 1; 118(9):2530-40. doi: 10.1182/blood-2011-01-330357. Epub    2011 Jul. 18, Antigenic modulation limits the effector cell    mechanisms employed by type I anti-CD20 monoclonal antibodies.

1.-15. (canceled)
 16. A method of treating an FcγRIIb-expressing cancerin a subject, the method comprising administering to the subject acomposition that comprises an engineered monomeric IgA antibody in anamount sufficient to treat the FcγRIIb-expressing cancer in the subject,wherein the monomeric IgA antibody comprises an IgA heavy chain constantregion, and does not comprise any IgG constant region domain; andwherein the monomeric IgA antibody induces caspase-independentprogrammed cell death (PCD) of FcγRIIb-expressing cancer cells to agreat extent relative to caspase-independent programmed cell death (PCD)of FcγRIIb-expressing cancer cells induced by a corresponding IgGantibody that has the same variable region complementarity determiningregions (CDRs) as the engineered monomeric IgA antibody, as determinedin an in vitro assay comprising: (i) contacting a first population ofcancer cells that express FcγRIIb with the engineered monomeric IgAantibody, (ii) contacting a second population of cancer cells thatexpress FcγRIIb with the corresponding IgG antibody, (iii) contactingthe first population of cancer cells and the second population of cancercells with Annexin V, and (iv) comparing an amount of fluorescenceexhibited by the first population of cancer cells and the secondpopulation of cancer cells using a FACS cell sorter, wherein a greateramount of fluorescence indicates a greater amount of PCD.
 17. The methodof claim 16, wherein the engineered monomeric IgA antibody is an IgA1antibody.
 18. The method of claim 16, wherein the engineered monomericIgA antibody is an IgA2 antibody.
 19. The method of claim 16, whereinthe FcγRIIb-expressing cancer is resistant to the corresponding IgGantibody.
 20. The method of claim 16, wherein the IgA heavy chainconstant region comprises 1-5 substitutions, insertions, or deletions,relative to the amino acid sequence of SEQ ID NO:4.
 21. The method ofclaim 16, wherein the IgA heavy chain constant region comprises 1-5substitutions, insertions, or deletions, relative to the amino acidsequence of SEQ ID NO:5.
 22. The method of claim 16, wherein the IgAheavy chain constant region comprises an IgA2 hinge.
 23. The method ofclaim 16, wherein the engineered monomeric IgA antibody inducesantibody-dependent cell-mediated cytotoxicity (ADCC) of the cancer cell.24. The method of claim 23, wherein the engineered monomeric IgAantibody facilitates increased ADCC of the cancer cell, relative to thecorresponding IgG antibody.
 25. The method of claim 16, wherein theengineered monomeric IgA antibody induces antibody-dependentcell-mediated phagocytosis (ADCP) of the cancer cell.
 26. The method ofclaim 16, wherein the PCD is neutrophil-mediated PCD.
 27. The method ofclaim 16, wherein the PCD is macrophage-mediated PCD.
 28. The method ofclaim 16, wherein the engineered monomeric IgA antibody binds to FcαRI.29. The method of claim 16, wherein the engineered monomeric IgAantibody binds to an antigen selected from the group consisting of:CD20, EGFR, and Her2.
 30. The method of claim 16, wherein theFcγRIIb-expressing cancer is a solid tumor cancer.
 31. The method ofclaim 16, wherein the FcγRIIb-expressing cancer is lymphoma.
 32. Themethod of claim 16, wherein the subject is a human.
 33. The method ofclaim 16, wherein the subject is a child.